Metal chelate containing compositions for use in chemiluminescent assays

ABSTRACT

Compositions are disclosed comprising (a) a metal chelate wherein the metal is selected from the group consisting of europium, terbium, dysprosium, samarium osmium and ruthenium in at least a hexacoordinated state and (b) a compound having a double bond substituted with two aryl groups, an oxygen atom and an atom selected from the group consisting of oxygen, sulfur and nitrogen wherein one of the aryl groups is electron donating with respect to the other. Such composition is preferably incorporated in a latex particulate material. Methods and kits are also disclosed for determining an analyte in a medium suspected of containing the analyte. The methods and kits employ as one component a composition as described above.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of pending application Ser. No. 08/156,181, filedNov. 22, 1993, now U.S. Pat. No. 5,578,498 which in turn is acontinuation-in-part of application Ser. No. 07/704,569, filed May 22,1991, the disclosures of which are incorporated herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods, compositions and kits for determiningan analyte in a sample. In particular, this invention relates tocompositions that exhibit a high quantum yield chemiluminescence whenactivated by singlet oxygen, decay rapidly and emit at long wavelengths.

The clinical diagnostic field has seen a broad expansion in recentyears, both as to the variety of materials (analytes) that may bereadily and accurately determined, as well as the methods for thedetermination. Convenient, reliable and non-hazardous means fordetecting the presence of low concentrations of materials in liquids isdesired. In clinical chemistry these materials may be present in bodyfluids in concentrations below 10⁻¹² molar. The difficulty of detectinglow concentrations of these materials is enhanced by the relativelysmall sample sizes that can be utilized.

In developing an assay there are many considerations. One considerationis the signal response to changes in the concentration of analyte. Asecond consideration is the ease with which the protocol for the assaymay be carried out. A third consideration is the variation ininterference from sample to sample. Ease of preparation and purificationof the reagents, availability of equipment, ease of automation andinteraction with material of interest are some of the additionalconsiderations in developing a useful assay.

One broad category of techniques involves the use of a receptor whichcan specifically bind to a particular spacial and polar organization ofa labeled ligand as a function of the presence of an analyte. Theobserved effect of binding by the receptor will depend upon the label.In some instances the binding of the receptor merely provides for adifferentiation in molecular weight between bound and unbound labeledligand. In other instances the binding of the receptor will facilitateseparation of bound labeled ligand from free labeled ligand or it mayaffect the nature of the signal obtained from the label so that thesignal varies with the amount of receptor bound to labeled ligand. Afurther variation is that the receptor is labeled and the ligandunlabeled. Alternatively, both the receptor and ligand are labeled ordifferent receptors are labeled with two different labels, whereupon thelabels interact when in close proximity and the amount of ligand presentaffects the degree to which the labels of the receptor may interact.

There is a continuing need for new and accurate techniques that can beadapted for a wide spectrum of different ligands or be used in specificcases where other methods may not be readily adaptable.

Homogeneous immunoassays have previously been described for smallmolecules. These assays include SYVA's FRAT® assay, EMIT® assay, enzymechanneling immunoassay, and fluorescence energy transfer immunoassay(FETI); enzyme inhibitor immunoassays (Hoffman LaRoche and AbbottLaboratories): fluorescence polarization immunoassay (Dandlicker), amongothers. All of these methods have limited sensitivity, and only a fewincluding FETI and enzyme channeling, are suitable for largemultiepitopic analytes. Luminescent compounds, such as fluorescentcompounds and chemiluminescent compounds, find wide application in theassay field because of their ability to emit light. For this reason,luminescers have been utilized as labels in assays such as nucleic acidassays and immunoassays. For example, a member of a specific bindingpair is conjugated to a luminescer and various protocols are employed.The luminescer conjugate can be partitioned between a solid phase and aliquid phase in relation to the amount of analyte in a sample suspectedof containing the analyte. By measuring the luminescence of either ofthe phases, one can relate the level of luminescence observed to aconcentration of the analyte in the sample.

Particles, such as liposomes and erythrocyte ghosts, have been utilizedas carriers of encapsulated water soluble materials. For example,liposomes have been employed to encapsulate biologically active materialfor a variety of uses, such as drug delivery systems wherein amedicament is entrapped during liposome preparation and thenadministered to the patient-to be treated.

Particles, such as latex beads and liposomes, have also been utilized inassays. For example, in homogeneous assays an enzyme may be entrapped inthe aqueous phase of a liposome labelled with an antibody or antigen.The liposomes are caused to release the enzyme in the presence of asample and complement. Antibody- or antigen-labelled liposomes, havingwater soluble fluorescent or non-fluorescent dyes encapsulated within anaqueous phase or lipid soluble dyes dissolved in the lipid bilayer ofthe lipid vesicle or in latex beads, have also been utilized to assayfor analytes capable of entering into an immunochemical reaction withthe surface bound antibody or antigen. Detergents have been used torelease the dyes from the aqueous phase of the liposomes.

2. Brief Description of the Related Art

White, et al. (White), discuss “Chemically Produced Excited States.Energy Transfer, Photochemical Reactions, and Light Emission” in J. Am.Chem. Soc., 93, 6286 (1971).

McCapra, et al. (McCapra), disclose “Metal Catalysed Light Emission froma Dioxetan” in Tetrahedron Letters, 23:49, 5225-5228 (1982).

Wildes, et al. (Wildes), discuss “The Dioxetane-SensitizedChemiluminescence of Lanthanide Chelates. A Chemical Source of‘Monochromatic’ Light” in J. Am. Chem. Soc., 93(23), 6286-6288 (1971).

Handley, et al. (Handley), disclose “Effects of Heteroatom Substituentson the Properties of 1,2-Dioxetanes” in Tetradron Letters, 26, 3183(1985).

Zaklika, et al. (Zaklika), discuss “Substituent Effects on theDecompositon of 1,2-Dioxetanes” in J. Am. Chem. Soc., 100, 4916 (1978).

European Patent Application No. 0,345,776 (McCapra) discloses specificbinding assays that utilize a sensitizer as a label. The sensitizersinclude any moiety which, when stimulated by excitation with radiationof one or more wavelengths or other chemical or physical stimulus (e.g.,electron transfer, electrolysis, electroluminescence or energy transfer)will achieve an excited state which (a) upon interaction with molecularoxygen will produce singlet molecular oxygen, or (b) upon interactionwith a leuco dye will assume a reduced form that can be returned to itsoriginal unexcited state by interaction with molecular oxygen resultingin the production of hydrogen peroxide. Either interaction with theexcited sensitizer will, with the addition of reagents, produce adetectible signal.

European Patent Application No. 0,070,685 (Heller, et al. I) describes ahomogeneous nucleic acid hybridization diagnostic by non-radiativeenergy transfer.

A light-emitting polynucleotide hybridization diagnostic method isdescribed in European Patent Application No. 0,070,687 (Heller, et al.II).

SUMMARY OF THE INVENTION

One aspect of the present invention is directed to compositionscomprising (a) a metal chelate comprising a metal selected from thegroup consisting of europium, terbium, dysprosium, samarium, osmium andruthenium in at least a hexacoordinated state and (b) a compound havinga structural portion that is a double bond substituted with two arylgroups, an oxygen atom and an atom selected from the group consisting ofoxygen, sulfur and nitrogen. The aryl groups are characterized in thatone is electron donating with respect to the other. Preferably, thecomposition is incorporated in a latex particulate material.

Another aspect of the present invention is a compound of the formula:

wherein X′ is S or NR′ wherein R′ is alkyl or aryl and D and D′ areindependently selected from the group consisting of alkyl and alkylradical.

Another aspect of the present invention is a composition comprising alatex having incorporated therein a compound of the formula:

wherein X″ is O, S or NR″ wherein R″ is alkyl or aryl, n is 1 to 4, andAr and Ar′ are independently aryl wherein one of Ar or Ar′ is electrondonating with respect to the other and Y is hydrogen or an organicradical consisting of atoms selected from the group consisting of C, O,N, S, and P and m is 0 to 2.

Another aspect of the present invention is a composition comprising alatex having incorporated therein Compound 1.

Another aspect of the present invention is a method for determining ananalyte which comprises (a) providing in combination (1) a mediumsuspected of containing an analyte, (2) a photosensitizer capable in itsexcited state of activating oxygen to a singlet state, where thephotosensitizer is associated with a specific binding pair (sbp) member,and (3) one of the above-mentioned compositions incorporated into alatex particulate material having bound thereto an sbp member, (b)treating the combination with light to excite the photosensitizer, and(c) examining the combination for the amount of luminescence emittedtherefrom. The amount of luminescence is related to the amount ofanalyte in the medium.

Another aspect of the present invention is a kit comprising in packagedcombination: (1) a composition comprising a suspendible latex particlecomprising one of the above-mentioned compounds and (2) aphotosensitizer. The particle has bound thereto a specific binding pair(sbp) member. The photosensitizer is capable in its excited state ofactivating oxygen to its singlet state.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention is directed to chemiluminescent compositions thatupon activation by singlet oxygen exhibit chemiluminescent emission thatrapidly decays, generally having a half life of 0.5 seconds to 30minutes, preferably 0.5 to 30 seconds, usually less than twenty seconds.In addition, the present chemiluminescent compositions can exhibit highchemiluminescent quantum yield upon activation by singlet oxygen,generally 0.1 to 0.9, usually 0.1 to 0.6, preferably 0.2 to 0.4. Thechemiluminescent light emitted by the metal chelate after activation inthe present compositions generally has a wave length of about 550 to 700nm, usually greater than 600 nm. The chemiluminescent compositions ofthe present invention are particularly useful in luminescent assays. Forexample, the long wavelength emission avoids interference from serumabsorption in assays on blood or serum samples. The high quantum yieldimproves detectibility and the short lifetime further improvesdetectibility by causing all the light that is emitted to be deliveredin a short pulse rather than over an extended period of time. This canprovide higher light intensity at lower quantum yields.

The quantum yield of chemiluminescence of the present chemiluminescentcompositions, when activated by singlet oxygen, is generally about 10 to100 fold greater, preferably, 10 to 50 fold greater, than that observedupon irradiation of the components of the composition separately.Furthermore, the rate of decay of chemiluminescence is significantlyenhanced with some of the present compositions. These properties renderthe present compositions extremely useful in assays for thedetermination of analytes.

Before proceeding further with a description of the specific embodimentsof the present invention, a number of terms will be defined anddescribed in detail.

Metal ligand—a compound in which two or more atoms of the same moleculecan coordinate with a metal to form a metal chelate. The metal chelatesthat form part of the compositions of the present invention comprise ametal selected from the group consisting of europium, terbium,dysprosium, samarium, osmium and ruthenium. One of the above metals iscoordinated with one or more metal ligands, which may be, for example,3-(2-thienoly)-1,1,1-trifluoroacetone (TTA),3-benzoyl-1,1,1-trifluoroacetone (BFTA),3-naphthoyl-1,1,1-trifluoroacetone (NPPTA),2,2-dimethyl-4-perfluorobutyoyl-3-butanone (fod), 2,2′-dipyridyl (bpy),phenanthroline(phen), salicylic acid, phenanthroline carboxylic acid,bipyridyl carboxylic acid, aza crown ethers trioctylphosphine oxide, azacryptands, and so forth. Usually, the metal in the metal chelate is atleast hexacoordinated, but may be octacoordinated or more highlycoordinated depending on the metal ligands. The metal chelate will beuncharged, thus the number of acidic groups provided by its ligands willequal the oxidation state of the metal. Usually, the metal ligands willbe relatively hydrophobic so as to impart solubility of the metalchelate in non-polar solvents. Rare earth metals will usually have anoxidation state of three, ruthenium will have an oxidation state of twoand osmium will have an oxidation state of two. Examplary of such metalchelates, by way of illustration and not limitation, is as follows:

One TTA in 3(a) or 3(b) can be placed by one of the following:

wherein DPP (Diphenylphenanthroline) in 3(b) can be replaced by one ofthe following:

Two TTA's in 3(a) and 3(b) can be independently replaced by compoundsselected from the following:

Three TTA's can be independently replaced by compounds selected from thefollowing:

Many of these metal ligands and metal chelates are known in the art andmany are commercially available. In general, metal chelates can beprepared from metal ligands by combining the metal chloride with thedesired ratio of metal ligand molecules in an organic solvent such as,e.g., acetonitrile and sufficient base, e.g., pyridine, to take up thereleased hydrochloric acid. For example, metal chelates can be preparedby a procedure such as that described by Shinha, A. P., “Fluorescencesand laser action in rare earth chelates,” Spectroscopy InorganicChemistry, Vol 2, (1971), 255-288.

Aryl group—an organic radical derived from an aromatic hydrocarbon bythe removal of one atom and containing one or more aromatic rings,usually one to four aromatic rings, which are generally five- orsix-member rings such as, e.g., phenyl (from benzene), naphthyl (fromnaphthalene), biphenylenyl, azulenyl, anthryl, phenanthrenyl, pyridyl,indolyl, benzofuranyl, benzothiophenyl, quinolinyl, isoquinolinyl,carbazolyl, acridinyl, imidazolyl, thiazolyl, pyrazinyl, pyrimidinyl,purinyl, pteridinyl, etc.

Aralkyl—an organic radical having an alkyl group to which is attached anaryl group, e.g., benzyl, phenethyl, 3-phenylpropyl, 1-naphthylethyl,etc.

Electron donating group—a substituent which when bound to a molecule iscapable of polarizing the molecule such that the electron donating groupbecomes electron poor and positively charged relative to another portionof the molecule, i.e., has reduced electron density. Such groups may be,by way of illustration and not limitation, amines, ethers, thioethers,phosphines, hydroxy, oxyanions, mercaptans and their anions, sulfides,etc.

Alkyl—a monovalent branched or unbranched radical derived from analiphatic hydrocarbon by removal of one H atom; includes both loweralkyl and upper alkyl.

Alkyl radical—a substituent formed from two or more alkyl groups, whichmay be independently lower or upper alkyl groups, linked together by afunctionality such as an ether, including thioether, an amide, an esterand the like.

Lower Alkyl—alkyl containing from 1 to 5 carbon atoms such as, e.g.,methyl, ethyl, propyl, butyl, isopropyl, isobutyl, pentyl, isopentyl,etc.

Upper Alkyl—alkyl containing more than 6 carbon atoms, usually 6 to 20carbon atoms, such as, e.g., hexyl, heptyl, octyl, etc.

Alkylidene—a divalent organic radical derived from an aliphatichydrocarbon, such as, for example, ethylidene, in which 2 hydrogen atomsare taken from the same carbon atom.

Substituted—means that a hydrogen atom of a molecule has been replacedby another atom, which may be a single atom such as a halogen, etc., orpart of a group of atoms forming a functionality such as a substituenthaving from 1 to 50 atoms (other than the requisite hydrogen atomsnecessary to satisfy the valencies of such atoms), which atoms areindependently selected from the group consisting of carbon, oxygen,nitrogen, sulfur and phosphorus, and which may or may not be bound toone or more metal atoms.

Analyte—the compound or composition to be detected. The analyte can becomprised of a member of a specific binding pair (sbp) and may be aligand, which is monovalent (monoepitopic) or polyvalent (polyepitopic),usually antigenic or haptenic, and is a single compound or plurality ofcompounds which share at least one common epitopic or determinant site.The analyte can be a part of a cell such as bacteria or a cell bearing ablood group antigen such as A, B, D, etc., or an HLA antigen or amicroorganism, e.g., bacterium, fungus, protozoan, or virus.

The polyvalent ligand analytes will normally be poly(amino acids), i.e.,polypeptides and proteins, polysaccharides, nucleic acids, andcombinations thereof. Such combinations include components of bacteria,viruses, chromosomes, genes, mitochondria, nuclei, cell membranes andthe like.

For the most part, the polyepitopic ligand analytes to which the subjectinvention can be applied will have a molecular weight of at least about5,000, more usually at least about 10,000. In the poly(amino acid)category, the poly(amino acids) of interest will generally be from about5,000 to 5,000,000 molecular weight, more usually from about 20,000 to1,000,000 molecular weight; among the hormones of interest, themolecular weights will usually range from about 5,000 to 60,000molecular weight.

A wide variety of proteins may be considered as to the family ofproteins having similar structural features, proteins having particularbiological functions, proteins related to specific microorganisms,particularly disease causing microorganisms, etc. Such proteins include,for example, immuloglobulins, cytokines, enzymes, hormones, cancerantigens, nutritional markers, tissue specific antigens, etc.

The following are classes of proteins related by structure: protamines,histones, albumins, globulins, scleroproteins, phosphoproteins,mucoproteins, chromoproteins, lipoproteins, nucleoproteins,glycoproteins, T-cell receptors, proteoglycans, HLA, unclassifiedproteins, e.g., somatotropin, prolactin, insulin, pepsin, proteins foundin the human plasma such as blood clotting factors, other polymericmaterials such as mucopolysaccharides and polysaccharides,microorganisms such as bacteria, viruses and fungi.

The monoepitopic ligand analytes will generally be from about 100 to2,000 molecular weight, more usually from 125 to 1,000 molecular weight.The analytes include drugs, metabolites, pesticides, pollutants, and thelike. Included among drugs of interest are the alkaloids, steroids,steroid mimetic substances, lactams, aminoalkylbenzenes,benzheterocyclics, purines, those derived from marijuana, hormones,vitamins, prostaglandins, tricyclic antidepressants, anti-neoplastics,antibiotics, nucleosides and nucleotides, miscellaneous individual drugswhich include methadone, meprobamate, serotonin, meperidine, lidocaine,procainamide, acetylprocainamide, propranolol, griseofulvin, valproicacid, butyrophenones, antihistamines, chloramphenicol, anticholinergicdrugs, such as atropine, metabolites related to diseased states includespermine, galactose, phenylpyruvic acid, and porphyrin Type 1,aminoglycosides, polyhalogenated biphenyls, phosphate esters,thiophosphates, carbamates, polyhalogenated sulfenamides.

For receptor analytes, the molecular weights will generally range from10,000 to 2×10⁸, more usually from 10,000 to 10⁶. For immunoglobulins,IgA, IgG, IgE and IgM, the molecular weights will generally vary fromabout 160,000 to about 10⁶. Enzymes will normally range from about10,000 to 1,000,000 in molecular weight. Natural receptors vary widely,generally-being at least about 25,000 molecular weight and may be 10⁶ orhigher molecular weight, including such materials as avidin, DNA, RNA,thyroxine binding globulin, thyroxine binding prealbumin, transcortin,etc.

The term analyte further includes polynucleotide analytes such as thosepolynucleotides defined below. These include m-RNA, r-RNA, t-RNA, DNA,DNA-RNA duplexes, etc. The term analyte also includes receptors that arepolynucleotide binding agents, such as, for example, restrictionenzymes, activators, repressors, nucleases, polymerases, histones,repair enzymes, chemotherapeutic agents, and the like.

The analyte may be a molecule found directly in a sample such as a bodyfluid from a host. The sample can be examined directly or may bepretreated to render the analyte more readily detectible. Furthermore,the analyte of interest may be determined by detecting an agentprobative of the analyte of interest such as a specific binding pairmember complementary to the analyte of interest, whose presence will bedetected only when the analyte of interest is present in a sample. Thus,the agent probative of the analyte becomes the analyte that is detectedin an assay. The body fluid can be, for example, urine, blood, plasma,serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears,mucus, and the like.

Member of a Specific Binding Pair (“sbp member”)—one of two differentmolecules, having an area on the surface or in a cavity whichspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of the other molecule. Themembers of the specific binding pair are referred to as ligand andreceptor (antiligand). These will usually be members of an immunologicalpair such as antigen-antibody, although other specific binding pairssuch as biotin-avidin, hormones-hormone receptors, nucleic acidduplexes, IgG-protein A, polynucleotide pairs such as DNA-DNA, DNA-RNA,and the like are not immunological pairs but are included in theinvention and the definition of sbp member.

Polynucleotide—a compound or composition which is a polymeric nucleotidehaving in the natural state about 50 to 500,000 or more nucleotides andhaving in the isolated state about 15 to 50,000 or more nucleotides,usually about 15 to 20,000 nucleotides, more frequently 15 to 10,000nucleotides. The polynucleotide includes nucleic acids from any sourcein purified or unpurified form, naturally occurring or syntheticallyproduced, including DNA (dsDNA and ssDNA) and RNA, usually DNA, and maybe t-RNA, m-RNA, r-RNA, mitochondrial DNA and RNA, chloroplast DNA andRNA, DNA-RNA hybrids, or mixtures thereof, genes, chromosomes, plasmids,the genomes of biological material such as microorganisms, e.g.,bacteria, yeasts, viruses, viroids, molds, fungi, plants, animals,humans, and fragments thereof, and the like.

Ligand—any organic compound for which a receptor naturally exists or canbe prepared.

Ligand Analog—a modified ligand, an organic radical or analyte analog,usually of a molecular weight greater than 100, which can compete withthe analogous ligand for a receptor, the modification providing means tojoin a ligand analog to another molecule. The ligand analog will usuallydiffer from the ligand by more than replacement of a hydrogen with abond which links the ligand analog to a hub or label, but need not. Theligand analog can bind to the receptor in a manner similar to theligand. The analog could be, for example, an antibody directed againstthe idiotype of an antibody to the ligand.

Receptor (“antiligand”)—any compound or composition capable ofrecognizing a particular spatial and polar organization of a molecule,e.g., epitopic or determinant site. Illustrative receptors includenaturally occurring receptors, e.g., thyroxine binding globulin,antibodies, enzymes, Fab fragments, lectins, nucleic acids, protein A,complement component C1q, and the like.

Specific binding—the specific recognition of one of two differentmolecules for the other compared to substantially less recognition ofother molecules. Generally, the molecules have areas on their surfacesor in cavities giving rise to specific recognition between the twomolecules. Exemplary of specific binding are antibody-antigeninteractions, enzyme—substrate interactions, polynucleotideinteractions, and so forth.

Non-specific binding—non-covalent binding between molecules that isrelatively independent of specific surface structures. Non-specificbinding may result from several factors including hydrophobicinteractions between molecules.

Antibody—an immunoglobulin which specifically binds to and is therebydefined as complementary with a particular spatial and polarorganization of another molecule. The antibody can be monoclonal orpolyclonal and can be prepared by techniques that are well known in theart such as immunization of a host and collection of sera (polyclonal)or by preparing continuous hybrid cell lines and collecting the secretedprotein (monoclonal), or by cloning and expressing nucleotide sequencesor mutagenized versions thereof coding at least for the amino acidsequences required for specific binding of natural antibodies.Antibodies may include a complete immunoglobulin or fragment thereof,which immunoglobulins include the various classes and isotypes, such asIgA, IgD, IgE, IgG1, IgG2a, IgG2b and IgG3, IgM, etc. Fragments thereofmay include Fab, Fv and F(ab′)₂, Fab′, and the like. In addition,aggregates, polymers, and conjugates of immunoglobulins or theirfragments can be used where appropriate so long as binding affinity fora particular molecule is maintained.

A substituent having from 1 to 50 atoms (other than the requisitehydrogen atoms necessary to satisfy the valencies of such atoms), whichatoms are independently selected from the group consisting of carbon,oxygen, nitrogen, sulfur and phosphorus—an organic radical; the organicradical has 1 to 50 atoms other than the requisite number of hydrogenatoms necessary to satisfy the valencies of the atoms in the radical.Generally, the predominant atom is carbon (C) but may also be oxygen(O), nitrogen (N), sulfur (S), phosphorus (P), wherein the O, N, S, orP, if present, are bound to carbon or one or more of each other or tohydrogen or a metal atom to form various functional groups, such as, forexample, carboxylic acids, alcohols, thiols, carboxamides, carbamates,carboxylic acid esters, sulfonic acids, sulfonic acid esters, phosphoricacids, phosphoric acid esters, ureas, carbamates, phosphoramides,sulfonamides, ethers, sulfides, thioethers, olefins, acetylenes, amines,ketones, aldehydes, nitrites, and the like. Illustrative of such organicradicals or groups, by way of illustration and not limitation, arealkyl, alkylidine, aryl, aralkyl, and alkyl, aryl, and aralkylsubstituted with one or more of the aforementioned functionalities.

Linking group—the covalent linkage between molecules. The linking groupwill vary depending upon the nature of the molecules, i.e.,photosensitizer, chemiluminescent compound, sbp member or moleculeassociated with or part of a particle, being linked. Functional groupsthat are normally present or are introduced on a photosensitizer orchemiluminescent compound will be employed for linking these materialsto an sbp member or a particle such as a latex particle.

For the most part, carbonyl functionalities will find use, bothoxocarbonyl, e.g., aldehyde and non-oxocarbonyl (including nitrogen andsulfur analogs) e.g., carboxy, amidine, amidate, thiocarboxy andthionocarboxy.

Alternative functionalities of oxo include active halogen, diazo,mercapto, olefin, particularly activated olefin, amino, phosphoro andthe like. A description of linking groups may be found in U.S. Pat. No.3,817,837, which disclosure is incorporated herein by reference.

Common functionalities in forming a covalent bond between the linkinggroup and the molecule to be conjugated are alkylamine, amidine,thioamide, ether, urea, thiourea, guanidine, azo, thioether andcarboxylate, sulfonate, and phosphate esters, amides and thioesters.

For the most part, the photosensitizer and chemilumenescent compoundwill have a non-oxocarbonyl group including nitrogen and sulfur analogs,a phosphate group, an amino group, alkylating agent such as halo ortosylalkyl, oxy (hydroxyl or the sulfur analog, mercapto) oxocarbonyl(e.g., aldehyde or ketone), or active olefin such as a vinyl sulfone orα, β-unsaturated ester. These functionalities will be linked to aminegroups, carboxyl groups, active olefins, alkylating agents, e.g.,bromoacetyl. Where an amine and carboxylic acid or its nitrogenderivative or phophoric acid are linked, amides, amidines andphosphoramides will be formed. Where mercaptan and activated olefin arelinked, thioethers will be formed. Where a mercaptan and an alkylatingagent are linked, thioethers will be formed. Where aldehyde and an amineare linked under reducing conditions, an alkylamine will be formed.Where a carboxylic acid or phosphate acid and an alcohol are linked,esters will be formed.

Photosensitizer—a sensitizer for generation of singlet oxygen usually byexcitation with light. The photosensitizer can be photoactivatable(e.g., dyes and aromatic compounds) or chemiactivated (e.g., enzymes andmetal salts). When excited by light the photosensitizer is usually acompound comprised of covalently bonded atoms, usually with multipleconjugated double or triple bonds. The compound should absorb light inthe wavelength range of 200-1100 nm, usually 300-1000 nm, preferably450-950 nm, with an extinction coefficient at its absorbance maximumgreater than 500 M⁻¹ cm⁻¹, preferably at least 5000 M¹ cm⁻¹, morepreferably at least 50,000 M⁻¹ cm⁻¹ at the excitation wavelength. Thelifetime of an excited state produced following absorption of light inthe absence of oxygen will usually be at least 100 nsec, preferably atleast 1 msec. In general, the lifetime must be sufficiently long topermit energy transfer to oxygen, which will normally be present atconcentrations in the range of 10⁵ to 10⁻¹M depending on the medium. Thesensitizer excited state will usually have a different spin quantumnumber (S) than its ground state and will usually be a triplet (S=1)when, as is usually the case, the ground state is a singlet (S=0).Preferably, the sensitizer will have a high intersystem crossing yield.That is, photoexcitation of a sensitizer will produce the long livedstate (usually triplet) with an efficiency of at least 10%, desirably atleast 40%, preferably greater than 80%. The photosensitizer will usuallybe at most weakly fluorescent under the assay conditions (quantum yieldusually less that 0.5, preferably less that 0.1).

Photosensitizers that are to be excited by light will be relativelyphotostable and will not react efficiently with singlet oxygen. Severalstructural features are present in most useful sensitizers. Mostsensitizers have at least one and frequently three or more conjugateddouble or triple bonds held in a rigid, frequently aromatic structure.They will frequently contain at least one group that acceleratesintersystem crossing such as a carbonyl or imine group or a heavy atomselected from rows 3-6 of the periodic table, especially iodine orbromine, or they may have extended aromatic structures. Typicalsensitizers include acetone, benzophenone, 9-thioxanthone, eosin,9,10-dibromoanthracene, methylene blue, metalloporphyrins, such ashematoporphyrin, phthalocyanines, chlorophylls, rose bengal,buckminsterfullerene, etc., and derivatives of these compounds havingsubstituents of 1 to 50 atoms for rendering such compounds morelipophilic or more hydrophilic and/or as attaching groups forattachment, for example, to an sbp member. Examples of otherphotosensitizers that may be utilized in the present invention are thosethat have the above properties and are enumerated in N. J. Turro,“Molecular Photochemistry”, page 132, W. A. Benjamin Inc., N.Y. 1965.

The photosensitizers are preferably relatively non-polar to assuredissolution into a lipophilic member when the photosensitizer isincorporated in an oil droplet, liposome, latex particle, etc.

The photosensitizers useful in this invention are also intended toinclude other substances and compositions that can produce singletoxygen with or, less preferably, without activation by an external lightsource. Thus, for example, molybdate (MoO₄ ⁼) salts and chloroperoxidaseand myeloperoxidase plus bromide or chloride ion (Kanofsky, J. Biol.Chem. (1983) 259 5596) have been shown to catalyze the conversion ofhydrogen peroxide to singlet oxygen and water. Either of thesecompositions can, for example, be included in particles to which isbound an sbp member and used in the assay method wherein hydrogenperoxide is included as an ancillary reagent, chloroperoxidase is boundto a surface and molybdate is incorporated in the aqueous phase of aliposome. Also included within the scope of the invention asphotosensitizers are compounds that are not true sensitizers but whichon excitation by heat, light, or chemical activation will release amolecule of singlet oxygen. The best known members of this class ofcompounds includes the endoperoxides such as1,4-biscarboxyethyl-1,4-naphthalene endoperoxide,9,10-diphenylanthracene-9,10-endoperoxide and 5,6,11,12-tetraphenylnaphthalene 5,12-endoperoxide. Heating or direct absorption of light bythese compounds releases singlet oxygen.

Support or Surface—a surface comprised of a porous or non-porous waterinsoluble material. The surface can have any one of a number of shapes,such as strip, rod, particle, including bead, and the like. The surfacecan be hydrophilic or capable of being rendered hydrophilic and includesinorganic powders such as silica, magnesium sulfate, and alumina;natural polymeric materials, particularly cellulosic materials andmaterials derived from cellulose, such as fiber containing papers, e.g.,filter paper, chromatographic paper, etc.; synthetic or modifiednaturally occurring polymers, such as nitrocellulose, cellulose acetate,poly (vinyl chloride), polyacrylamide, cross linked dextran, agarose,polyacrylate, polyethylene, polypropylene, poly(4-methylbutene),polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon,polyvinyl butyrate), etc.; either used by themselves or in conjunctionwith other materials; glass available as Bioglass, ceramics, metals, andthe like. Natural or synthetic assemblies such as liposomes,phospholipid vesicles, and cells can also be employed.

Binding of sbp members to the support or surface may be accomplished bywell-known techniques, commonly available in the literature. See, forexample, “Immobilized Enzymes,” Ichiro Chibata, Halsted Press, New York(1978) and Cuatrecasas, J. Biol. Chem., 245:3059 (1970).

Particles—particles of at least about 20 nm and not more than about 20microns, usually at least about 40 nm and less than about 10 microns,preferably from about 0.10 to 2.0 microns diameter, normally having avolume of less than 1 picoliter. The particle may be organic orinorganic, swellable or non-swellable, porous or non-porous, having anydensity, but preferably of a density approximating water, generally fromabout 0.7 to about 1.5 g/ml, preferably suspendible in water, andcomposed of material that can be transparent, partially transparent, oropaque. The particles may or may not have a charge, and when they arecharged, they are preferably negative. The particles may be solid (e.g.,polymer, metal, glass, organic and inorganic such as minerals, salts anddiatoms), oil droplets (e.g., hydrocarbon, fluorocarbon, silicon fluid),or vesicles (e.g., synthetic such as phospholipid or natural such ascells and organelles). The particles may be latex particles or otherparticles comprised of organic or inorganic polymers; lipid bilayers,e.g., liposomes, phospholipid vesicles; oil droplets; silicon particles;metal sols; cells; and dye crystallites.

The organic particles will normally be polymers, either addition orcondensation polymers, which are readily dispersible in the assaymedium. The organic particles will also be adsorptive orfunctionalizable so as to bind at their surface, either directly orindirectly, an sbp member and to bind at their surface or incorporatewithin their volume a photosensitizer or a chemiluminescent compound.

The particles can be derived from naturally occurring materials,naturally occurring materials which are synthetically modified andsynthetic materials. Natural or synthetic assemblies such as lipidbilayers, e.g., liposomes and non-phospholipid vesicles, are preferred.Among organic polymers of particular interest are polysaccharides,particularly cross-linked polysaccharides, such as agarose, which isavailable as Sepharose, dextran, available as Sephadex and Sephacryl,cellulose, starch, and the like; addition polymers, such as polystyrene,polyacrylamide, homopolymers and copolymers of derivatives of acrylateand methacrylate, particularly esters and amides having free hydroxylfunctionalities including hydrogels, and the like. Inorganic polymersinclude silicones, glasses, available as Bioglas, and the like. Soloinclude gold, selenium, and other metals. Particles may also bedispersed water insoluble dyes such as porphyrins, phthalocyanines,etc., which may also act as photosensitizers. Particles may also includediatoms, cells, viral particles, magnetosomes, cell nuclei and the like.

Where the particles are commercially available, the particle size may bevaried by breaking larger particles into smaller particles by mechanicalmeans, such as grinding, sonication, agitation, etc.

The particles will usually be polyfunctional or be capable of beingpolyfunctionalized or be capable of being bound to an sbp member,photosensitizer, or chemiluminescent compound through specific ornon-specific covalent or non-covalent interactions. A wide variety offunctional groups are available or can be incorporated. Exemplaryfunctional groups include carboxylic acids, aldehydes, amino groups,cyano groups, ethylene groups, hydroxyl groups, mercapto groups and thelike. When covalent attachment of a sbp member, chemiluminescentcompound or photosensitizer to the particle is employed, the manner oflinking is well known and is amply illustrated in the literature. Seefor example Cautrecasas, J Biol. Chem., 245:3059 (1970). The length of alinking group may vary widely, depending upon the nature of the compoundbeing linked, the nature of the particle, the effect of the distancebetween the compound being linked and the particle on the binding of sbpmembers and the analyte and the like.

The photosensitizer can be chosen to dissolve in or noncovalently bindto the surface of the particles. In this case these compounds willpreferably be hydrophobic to reduce their ability to dissociate from theparticle and thereby cause both compounds to associate with the sameparticle.

The number of photosensitizer or chemiluminescent molecules associatedwith each particle will on the average usually be at least one and maybe sufficiently high that the particle consists entirely ofphotosensitizer or chemiluminescer molecules. The preferred number ofmolecules will be selected empirically to provide the highest signal tobackground in the assay. In some cases this will be best achieved byassociating a multiplicity of different photosensitizer molecules toparticles. Usually, the photosensitizer or chemiluminescent compound tosbp member ratio in the particles should be at least 1, preferably atleast 100 to 1, and most preferably over 1,000 to 1.

Latex particles—“Latex” signifies a particulate water suspendible waterinsoluble polymeric material usually having particle dimensions of 20 nmto 20 mm, more preferably 100 to 1000 nm in diameter. The latex isfrequently a substituted polyethylene such as: polystyrene-butadiene,polyacrylamide polystyrene, polystyrene with amino groups, poly-acrylicacid, polymethacrylic acid, acrylonitrile-butadiene, styrene copolymers,polyvinyl acetate-acrylate, polyvinyl pyrridine, vinyl-chloride acrylatecopolymers, and the like. Non-crosslinked polymers of styrene andcarboxylated styrene or styrene functionalized with other active groupssuch as amino, hydroxyl, halo and the like are preferred. Frequently,copolymers of substituted styrenes with dienes such as butadiene will beused.

The association of the photosensitizer or chemiluminescent compound withlatex particles utilized in the present invention may involveincorporation during formation of the particles by polymerization butwill usually involve incorporation into preformed particles, usually bynoncovalent dissolution into the particles. Usually a solution of thechemiluminescent compound or sensitizer will be employed. Solvents thatmay be utilized include alcohols, including ethanol, ethylene glycol andbenzyl alcohol; amides such as dimethyl formamide, formamide, acetamideand tetramethyl urea and the like; sulfoxides such as dimethyl sulfoxideand sulfolane; and ethers such as carbitol, ethyl carbitol, dimethoxyethane and the like, and water. The use of solvents having high boilingpoints in which the particles are insoluble permits the use of elevatedtemperatures to facilitate dissolution of the compounds into theparticles and are particularly suitable. The solvents may be used singlyor in combination. Particularly preferred solvents for incorporatingphotosensitizer are those that will not quench the triplet excited stateof the photosensitizer either because of their intrinsic properties orbecause they can subsequently be removed from the particles by virtue oftheir ability to be dissolved in a solvent such as water that isinsoluble in the particles. Aromatic solvents are preferred, andgenerally solvents that are soluble in the particle. For incorporatingchemiluminescent compounds in particles a solvent should be selectedthat does not interfere with the luminescence because of their intrinsicproperties or ability to be removed from the particles. Frequently,aromatic solvents will also be preferred. Typical aromatic solventsinclude dibutylphthalate, benzonitrile, naphthonitrile,dioctylterephthalate, dichlorobenzene, diphenylether, dimethoxybenzene,etc.

Except when the photosensitizer or chemiluminescent compound is to becovalently bound to the particles, it will usually be preferable to useelectronically neutral photosensitizers or chemiluminescent compounds.It is preferable that the liquid medium selected does not soften thepolymer beads to the point of stickiness. A preferred techniquecomprises suspending the selected latex particles in a liquid medium inwhich the photosensitizer or chemiluminescent compound has at leastlimited solubility. Preferably, the concentrations of thephotosensitizer and chemiluminescent compound in the liquid media willbe selected to provide particles that have the highest efficiency ofsinglet oxygen formation and highest quantum yield of emission from thechemiluminescent compound in the media but less concentrated solutionswill sometimes be preferred. Distortion or dissolution of the particlesin the solvent can be prevented by adding a miscible cosolvent in whichthe particles are insoluble.

Generally, the temperature employed during the procedure will be chosento maximize the singlet oxygen formation ability of the photosensitizerlabeled particles and the quantum yield of the chemiluminescent compoundparticles with the proviso that the particles should not melt or becomeaggregated at the selected temperature. Elevated temperatures arenormally employed. The temperatures for the procedure will generallyrange from 20° C. to 200° C., more usually from 50° C. to 170° C. It hasbeen observed that some compounds that are nearly insoluble at roomtemperature, are soluble in, for example, low molecular weight alcohols,such as ethanol and ethylene glycol and the like, at elevatedtemperatures. Carboxylated modified latex particles have been shown totolerate low molecular weight alcohols at such temperatures.

An sbp member may be physically adsorbed on the surface of the latexparticle or may be covalently bonded to the particle. In cases whereinthe sbp member is only weakly bound to the surface of the latexparticle, the binding may in certain cases be unable to endureparticle-to-particle shear forces encountered during incubation andwashings. Therefore, it may be preferable to covalently bond sbp membersto the latex particles under conditions that will minimize adsorption.This may be accomplished by chemically activating the surface of thelatex. For example, the N-hydroxysuccinimide ester of surface carboxylgroups can be formed and the activated particles to reduce nonspecificbinding of assay components to the particle surface, are then contactedwith a linker having amino groups that will react with the ester groupsor directly with an sbp member that has an amino group. The linker willusually be selected to reduce nonspecific binding of assay components tothe particle surface and will preferably provide suitable functionalityfor both attachment to the latex particle and attachment of the sbpmember. Suitable materials include maleimidated aminodextran (MAD),polylysine, aminosaccharides, and the like. MAD can be prepared asdescribed by Hubert, et al., Proc. Natl. Acad. Sci., 75(7), 3143, 1978.

In one method, MAD is first attached to carboxyl-containing latexparticles using a water soluble carbodiimide, for example,1-(3-dimethylaminopropyl)-3-ethyl carbodiimide. The coated particles arethen equilibrated in reagents to prevent nonspecific binding. Suchreagents include proteins such as bovine gamma globulin (BGG), anddetergent, such as Tween 20, TRITON X-100 and the like. A sbp memberhaving a sulfhydryl group, or suitably modified to introduce asulfhydryl group, is then added to a suspension of the particles,whereupon a covalent bond is formed between the sbp member and the MADon the particles. Any excess unreacted sbp member can then be removed bywashing.

Chemiluminescent compound—compounds that form part of the compositionsof the present invention are enol ethers generally having the structuralportion selected from the group consisting of:

wherein Ar and Ar′ are independently aryl wherein one of Ar or Ar′,preferably Ar, is electron donating with respect to the other. This maybe achieved, for example, by the presence of one or more electrondonating groups in one of Ar or Ar′. The part of the above structuresrepresented by the broken lines are not critical to the presentinvention and may be any substituent as long as such substituent doesnot interfere with dioxetane formation and transfer of energy.Generally, the compounds are those of Compound 2 wherein, preferably, mis 0, and n is 1 to 3.

For the most part the compounds that form part of the presentcomposition have the structural portion:

wherein X is O, S or N wherein the valency of N is completed withhydrogen or an organic radical consisting of atoms selected from thegroup consisting of C, O, N, S, and P and Ar and Ar′ are independentlyaryl wherein one of Ar or Ar′ is electron donating with respect to theother.

The broken lines in the above structure signify that the ring can beindependently unsubstituted or substituted with a substituent havingfrom 1 to 50 atoms. In addition, the substituents may be taken togetherto form a ring such as, for example, aryl, which may in turn besubstituted with a substituent having from 1 to 50 atoms.

Exemplary enol ethers, by way of illustration and not limitation, areset forth in the following chart with reference to the followingstructure:

wherein Compounds 9-17 have the following moieties for X, Ar, and Ar′.

X Ar Ar′ * O

9 S

10 S

11 S

12 S

13 S

14 S

15

16

17 *Compounds 9-17

The chemiluminescent compounds undergo a chemical reaction with singletoxygen to form a metastable intermediate that can decompose with thesimultaneous or subsequent emission of light within the wavelength rangeof 250 to 1200 nm. Preferably, the intermediate decomposes spontaneouslywithout heating or addition of ancillary reagents following itsformation. However, addition of a reagent after formation of theintermediate or the use of elevated temperature to acceleratedecomposition will be required for some chemiluminescent compounds. Thechemiluminescent compounds are usually electron rich compounds thatreact with singlet oxygen, frequently with formation of dioxetanes ordioxetanones, such as those represented by the following structure wherethe substituents on the carbon (C) atoms are those present on thecorresponding olefin:

some of which decompose spontaneously, others by heating and/or bycatalysis usually by an electron rich energy acceptor, with the emissionof light. For some cases the dioxetane is spontaneously converted to ahydroperoxide whereupon basic pH is required to reform the dioxetane andpermit decomposition and light emission.

The chemiluminescent compounds of interest will generally emit atwavelengths above 300 nanometers and usually above 400 nm. Compoundsthat alone or together with a fluorescent molecule emit light atwavelengths beyond the region where serum components absorb light willbe of particular use in the present invention. The fluorescence of serumdrops off rapidly above 500 nm and becomes relatively unimportant above550 nm. Therefore, when the analyte is in serum, chemiluminescentcompounds that emit light above 550 nm, preferably above 600 nm are ofparticular interest. In order to avoid autosensitization of thechemiluminescent compound, it is preferable that the chemiluminescentcompounds do not absorb light used to excite the photosensitizer. Sinceit will generally be preferable to excite the sensitizer with lightwavelengths longer than 500 nm, it will therefore be desirable thatlight absorption by the chemiluminescent compound be very low above 500nm.

The chemiluminescent compounds of the present invention can be preparedin a number of different ways. In one approach a 2-thioethanolderivative is condensed with an appropriate diaryl substitutedalpha-hydroxy ketone (substituted benzoin) where one aryl is substitutedon the ketone carbon and the other is substituted on the carboncontaining the alpha-hydroxy group. The condensation reaction yields theappropriate enol ether directly. The above condensation can be carriedout in an inert solvent such as toluene. Usually, the temperature of thereaction is about 90-130° and the reaction is allowed to proceed for aperiod of 5-50 hours. Generally, the reaction is carried out at thereflux temperature of the combined reagents. The condensation is carriedout in the presence of a Lewis acid, for example, an acyl chloride,silyl chloride, stannous chloride, etc. The following reaction scheme isillustrative of the above-described method for preparing thechemiluminescent compounds of the present invention:

Another reaction scheme for preparing compounds in accordance with thepresent invention, particularly those containing an alkyl radical, isdepicted in the following schematic for synthesizing Compound 13:

SYNTHESIS OF C-26 THIOXENE (Compound 13)

In the above synthesis ethyl 5-bromovalerate is condensed withN-methylaniline to give 22 which is converted by Kilsmeier-Haaksynthesis (DMF/POCl₃) to aldehyde 23. Benzoin condensation of 23 withbenzaldehyde yields 24 which is hydrolyzed with potassium hydroxide andconverted to amide 25 with didecylamine and diphenylphosphoryl azide(DPPA). Conversion to Compound 13 was carried out by condensation withmercaptoethanol and trimethylsilylchloride.

Another approach for preparing compounds in accordance with the presentinvention, particularly involving regioselective synthesis is shown inthe following schematic for synthesizing Compound 14:

In the above synthesis reaction of p-nitrophenylacetic acid (27) withdecanal in the presence of pd/carbon and hydrogen gas at 100 psi givesdidecylamine 28, which is condensed with p-heptylbenzene to give ketone29. Bromine and trifluoroacetic acid are used to brominate 29 andbicarbonate converts the product to benzoin 30. Conversion to Compound14 is carried out by condensation with mercaptoethanol andtrimethylsilylchloride.

Ancillary Materials—Various ancillary materials will frequently beemployed in the assay in accordance with the present invention. Forexample, buffers will normally be present in the assay medium, as wellas stabilizers for the assay medium and the assay components.Frequently, in addition to these additives, proteins may be included,such as albumins, organic solvents such as formamide, quaternaryammonium salts, polycations such as dextran sulfate, surfactants,particularly non-ionic surfactants, binding enhancers, e.g.,polyalkylene glycols, or the like. When the photosensitizer is activatedchemically rather than by irradiation, hydrogen peroxide will often beincluded as an ancillary reagent. When it is desired to shift theemission wavelength of the chemiluminescent compound to longerwavelength or catalyse the decomposition of its oxygen-activated form, afluorescent molecule may be employed.

Wholly or Partially Sequentially—when the sample and various agentsutilized in the present invention are combined other than concomitantly(simultaneously), one or more may be combined with one or more of theremaining agents to form a subcombination. Each subcombination can thenbe subjected to one or more steps of the present method. Thus, each ofthe subcombinations can be incubated under conditions to achieve one ormore of the desired results.

One aspect of the present invention is directed to compositionscomprising (a) a metal chelate comprising a metal selected from thegroup consisting of europium, terbium, dysprosium, samarium, osmium andruthenium in at least a hexacoordinated state and (b) a compound havinga structural portion that is a double bond substituted with two arylgroups, an oxygen atom and an atom selected from the group consisting ofoxygen, sulfur and nitrogen. The aryl groups are characterized in thatone is electron donating with respect to the other. The composition ofthe present invention comprising a metal chelate and an olefiniccompound is generally in a medium that may be liquid or solid, usuallysolid particulate. The liquid medium is usually a high-boiling, waterimmisinble liquid such as one from the group comprising toluene, lipids,fluorocarbons, diphenylether, chlorobenzene, dioctylphthalate,dimethoxybenzene, mineral oil and triacylglycerides and the solidparticulate medium can be an organic polymer such as polystyrene,polymethylacrylate, polyacrylate, polyacrylamide, polyvinylchloride andcopolymers thereof, nylon and other polyamides, etc. Preferably, thecomposition is incorporated in a latex particulate material.

The metal chelate is present in an amount to maximize thechemiluminescent quantum yield and minimize the decay time ofchemiluminescence. Usually, the metal chelate is present at 0.2-500 mM,preferably 2-100 mM. In some circumstances, usually when the metalchelate is hexacoordinated, reduction in the decay time is accompaniedby a reduction in quantum yield and a balance must be reached betweenthese two effects. Accordingly, the concentration of the metal chelatein the composition should be adjusted to achieve such a balance. Theconcentration of the chemiluminescent compound in the composition isusually 0.1-500 mM, preferably 2-100 mM.

Preferred compounds of the present invention have the formula ofCompound 1. Representative of such compounds are Compounds 10-16.Particularly preferred compounds are those of the formula of Compound 1wherein X′ is S or NR′ wherein R′ is lower alkyl or aryl and D and D′are independently lower alkyl, preferably wherein X′ is S. Particularlypreferred compounds within the above are those wherein D and D′ aremethyl and R′ is methyl or phenyl, and a most preferred compound is onein which X′ is S and D and D′ are methyl. Compound 13 is one of the morepreferred of the above compounds.

One aspect of the present invention is a composition comprising a latexhaving incorporated therein Compound 2. Preferred compositions are thosewherein R′ is methyl or phenyl and wherein n is 1 or 2 and m is 0.Preferably, Ar is selected from the group consisting of 5-member and6-member aromatic and heteroaromatic rings. In a preferred embodiment Aris phenyl substituted with an electron donating group at a position ofthe phenyl that is meta or para to the carbon that is bonded to thedouble bond and Ar′ is phenyl. Exemplary compositions are thosecontaining a compound selected from the group consisting of Compounds9-16. The latex particles are usually suspendible and have an averagediameter of 0.04 to 4000 nanometer. For assays the particle will have anspb member bound to it and will have an average diameter of 100 to 1000micrometers.

Another embodiment of the present invention is a method for determiningan analyte. The method comprises (a) providing in combination (1) amedium suspected of containing an analyte, (2) a photosensitizer capablein its excited state of activating oxygen to a singlet state, thephotosensitizer associated with a specific binding pair (sbp) member,and (3) a suspendible latex particulate material comprising Compound 2.The particulate material has bound thereto an sbp member. Thecombination is treated with light, usually by irradiation, to excite thephotosensitizer, and is then examined for the amount of luminescenceemitted. The amount of such luminescence is related to the amount ofanalyte in the medium. The photosensitizer may be incorporated in asecond suspendible particulate material. Particularly usefulcompositions for determining an analyte in accordance with the presentinvention are those containing Compound 1.

In the assay protocol the components are provided in combination and thelight produced as a function of activation of oxygen by the sensitizerwill be a function of analyte concentration. Advantageously, the methodsof the present invention can be carried out without heating the mediumto produce light. Consequently, the assay of the present invention canbe conducted at a constant temperature.

The chemiluminescent compound may be bound to a sbp member that iscapable of binding directly or indirectly to the analyte or to an assaycomponent whose concentration is affected by the presence of theanalyte. The term “capable of binding directly or indirectly” means thatthe designated entity can bind specifically to the entity (directly) orcan bind specifically to a specific binding pair member or to a complexof two or more sbp members which is capable of binding the other entity(indirectly). Preferably, assays conducted in accordance with thepresent invention utilize one of the above compositions in a latexparticle. This latex particle has an sbp member generally capable ofbinding directly or indirectly to the analyte or a receptor for theanalyte. When the sbp members associated with the photosensitizer andthe chemiluminescent compound are both capable of binding to theanalyte, a sandwich assay protocol results. When one of the sbp membersassociated with the photosensitizer or chemiluminescent compound canbind both the analyte and an analyte analog, a competitive assayprotocol can result.

The photosensitizer is usually caused to activate the chemiluminescentcompound by irradiating the medium containing the above reactants. Themedium must be irradiated with light having a wavelength with energysufficient to convert the photosensitizer to an excited state andthereby render it capable of activating molecular oxygen to singletoxygen. The excited state for the photosensitizer capable of excitingmolecular oxygen is generally a triplet state which is more than about20, usually at least 23, Kcal/mol more energetic than thephotosensitizer ground state. Preferably, the medium is irradiated withlight having a wavelength of about 450 to 950 nm although shorterwavelengths can be used, for example, 230-950 nm. The luminescenceproduced may be measured in any convenient manner such asphotographically, visually or photometrically to determine the amountthereof, which is related to the amount of analyte in the medium.

Although it will usually be preferable to excite the photosensitizer byirradiation with light of a wavelength that is efficiently absorbed bythe photosensitizer, other means of excitation may be used as forexample by energy transfer from an excited state of an energy donor suchas a second photosensitizer. When a second photosensitizer is used,wavelengths of light can be used which are inefficiently absorbed by thephotosensitizer but efficiently absorbed by the second photosensitizer.The second photosensitizer may be bound to an assay component that isassociated, or becomes associated, with the first photosensitizer, forexample, bound to a surface or incorporated in the particle having thefirst photosensitizer. When a second photosensitizer is employed it willusually have a lowest energy singlet state at a higher energy than thelowest energy singlet state of the first photosensitizer.

The 632.6 nm emission line of a helium-neon laser is an inexpensivelight source for excitation. Photosensitizers with absorption maxima inthe region of about 620 to about 650 nm are compatible with the emissionline of a helium-neon laser and are, therefore, particularly useful inthe present invention.

The method and compositions of the invention may be adapted to mostassays involving sbp members such as ligand-receptor; e.g.,antigen-antibody reactions; polynucleotide binding assays, and so forth.The assays may be homogeneous or heterogeneous, competitive ornoncompetitive. The assay components, chemiluminescent compound andphotosensitizer, can be utilized in a number of ways with (1) a surface,when employed, (2) nucleic acid or receptor and (3) nucleic acid orligand. The association may involve covalent or non-covalent bonds.Those skilled in the art will be able to choose appropriate associationsdepending on the particular assay desired in view of the foregoing andthe following illustrative discussion.

In a homogeneous assay approach, the sample may be pretreated ifnecessary to remove unwanted materials. The reaction for anoncompetitive sandwich type assay can involve an sbp member, (e.g., anantibody, nucleic acid probe, receptor or ligand) complementary to theanalyte and associated with a chemiluminescent compound; aphotosensitizer associated with an sbp member, (e.g., antibody, nucleicacid probe, receptor or ligand) that is also complementary to theanalyte; the sample of interest; and any ancillary reagents required.Preferably, at least the chemiluminescent compound is incorporated inparticles to which an sbp member is attached. The photosensitizer may bedirectly attached to an sbp member or it may also be incorporated intoparticles. In a competitive protocol one sbp member can be a derivativeof the analyte and the other sbp member can be complementary to theanalyte, e.g., an antibody. In either protocol the components may becombined either simultaneously or wholly or partially sequentially. Theability of singlet oxygen produced by an activated photosensitizer toreact with the chemiluminescent compound is governed by the binding ofan sbp member to the analyte. Hence, the presence or amount of analytecan be determined by measuring the amount of light emitted uponactivation of the photosensitizer by irradiation, heating or addition ofa chemical reagent, preferably by irradiation. Both the binding reactionand detection of the extent thereof can be carried out in a homogeneoussolution without separation. This is an advantage of the presentinvention over prior art methods utilizing chemiluminescence.

In a heterogeneous assay approach, the assay components comprise asample suspected of containing an analyte which is an sbp member; an sbpmember bound to a support, which may be either a non-dispersible surfaceor a particle having associated with it one member of a group consistingof the chemiluminescent compound and the photosensitizer; and an sbpmember having the other member of the group associated with it whereinthe sbp members can independently, either directly or indirectly, bindthe analyte or a receptor for the analyte. These components aregenerally combined either simultaneously or wholly or partiallysequentially. The surface or particles are then separated from theliquid phase and either the separated phase or the liquid phase issubjected to conditions for activating the photosensitizer, usually byirradiating the particular phase in question, and measuring the amountof light emitted.

The binding reactions in an assay for the analyte will normally becarried out in an aqueous medium at a moderate pH, generally that whichprovides optimum assay sensitivity. Preferably, the activation of thephotosensitizer will also be carried out in an aqueous medium. However,when a separation step is employed, non-aqueous media such as, e.g.,acetonitrile, acetone, toluene, benzonitrile, etc. and aqueous mediawith pH values that are very high, i.e., greater than 10.0, or very low,i.e., less than 4.0, usually very high, can be used. As explained above,the assay can be performed either without separation (homogeneous) orwith separation (heterogeneous) of any of the assay components orproducts.

The aqueous medium may be solely water or may include from 0.01 to 80volume percent of a cosolvent but will usually include less than 40% ofa cosolvent when an sbp member is used that is a protein. The pH for themedium of the binding reaction will usually be in the range of about 4to 11, more usually in the range of about 5 to 10, and preferably in therange of about 6.5 to 9.5. When the pH is not changed during thegeneration of singlet oxygen the pH will usually be a compromise betweenoptimum binding of the binding members and the pH optimum for theproduction of signal and the stability of other reagents of the assay.When elevated pH's are required for signal production, a step involvingthe addition of an alkaline reagent can be inserted between the bindingreaction and generation of singlet oxygen and/or signal production.Usually the elevated pH will be greater than 10, usually 10-14. Forheterogenous assays non-aqueous solvents may also be used as mentionedabove, the main consideration being that the solvent not reactefficiently with singlet oxygen.

Various buffers may be used to achieve the desired pH and maintain thepH during an assay. Illustrative buffers include borate, phosphate,carbonate, tris, barbital and the like. The particular buffer employedis not critical to this invention, but in an individual assay one oranother buffer may be preferred.

Moderate temperatures are normally employed for carrying out the bindingreactions of proteinaceous ligands and receptors in the assay andusually constant temperature, preferably, 25° to 40°, during the periodof the measurement. Incubation temperatures for the binding reactionwill normally range from about 5° to 45° C., usually from about 15° to40° C., more usually 25° to 40° C. Where binding of nucleic acids occurin the assay, higher temperatures will frequently be used, usually 20°to 90°, more usually 35° to 75° C. Temperatures during measurements,that is, generation of singlet oxygen and light detection, willgenerally range from about 20° to 100°, more usually from about 25° to50° C., more usually 25° to 40° C.

The concentration of analyte which may be assayed will generally varyfrom about 10⁻⁴ to below 10⁻¹⁶ M, more usually from about 10⁻⁶ to 10⁻¹⁴M. Considerations, such as whether the assay is qualitative,semiquantitative or quantitative, the particular detection technique theconcentration of the analyte of interest, and the maximum desiredincubation times will normally determine the concentrations of thevarious reagents.

In competitive assays, while the concentrations of the various reagentsin the assay medium will generally be determined by the concentrationrange of interest of the analyte, the final concentration of each of thereagents will normally be determined empirically to optimize thesensitivity of the assay over the range. That is, a variation inconcentration of the analyte which is of significance should provide anaccurately measurable signal difference.

The concentration of the sbp members will depend on the analyteconcentration, the desired rate of binding, and the degree that the sbpmembers bind nonspecifically. Usually, the sbp members will be presentin at least the lowest expected analyte concentration, preferably atleast the highest analyte concentration expected, and for noncompetitiveassays the concentrations may be 10 to 10⁶ times the highest analyteconcentration but usually less than 10⁻⁴ M, preferably less than 10⁻⁶ M,frequently between 10⁻¹¹ and 10⁻⁷ M. The amount of photosensitizer orchemiluminescent compound associated with a sbp member will usually beat least one molecule per sbp member and may be as high as 10⁵, usuallyat least 10-10⁴ when the photosensitizer or chemiluminescent molecule isincorporated in a particle.

While the order of addition may be varied widely, there will be certainpreferences depending on the nature of the assay. The simplest order ofaddition is to add all the materials simultaneously. Alternatively, thereagents can be combined wholly or partially sequentially. When theassay is competitive, it will often be desirable to add the analyteanalog after combining the sample and an sbp member capable of bindingthe analyte. Optionally, an incubation step may be involved after thereagents are combined, generally ranging from about 30 seconds to 6hours, more usually from about 2 minutes to 1 hour before the sensitizeris caused to generate singlet oxygen and the light emission is measured.

In a particularly preferred order of addition, a first set of specificbinding pair members that are complementary to and/or homologous withthe analyte are combined with the analyte followed by the addition ofspecific binding pair members complementary to the first specificbinding pair members, each associated with a different member of thegroup consisting of a photosensitizer and a composition of the presentinvention. The assay mixture, or a separated component thereof, is thenirradiated and the light emission is measured.

In a homogeneous assay after all of the reagents have been combined,they can be incubated, if desired. Then, the combination is irradiatedand the resulting light emitted is measured. The emitted light isrelated to the amount of the analyte in the sample tested. The amountsof the reagents of the invention employed in a homogeneous assay dependon the nature of the analyte. Generally, the homogeneous assay of thepresent invention exhibits an increased sensitivity over known assayssuch as the EMIT® assay. This advantage results primarily because of theimproved signal to noise ratio obtained in the present method.

Another aspect of the present invention relates to kits useful forconveniently performing an assay method of the invention for determiningthe presence or amount of an analyte in a sample suspected of containingthe analyte. The kits comprise in packaged combination: (1) acomposition comprising a suspendible latex particle comprising acompound of the formula of Compound 2, preferably of Compound 1, wherethe particle can bind a specific binding pair (sbp) member, and (2) aphotosensitizer capable in its excited state of activating oxygen to itssinglet state. The photosensitizer can be part of a compositioncomprising a second suspendible particle comprising the photosensitizerwhere the second particle has bound thereto a sbp member or it may bedirectly bound to a sbp member. The kit can further include a writtendescription of a method in accordance with the present invention andinstructions for using the reagents of the kit in such method.

To enhance the versatility of the subject invention, the reagents can beprovided in packaged combination, in the same or separate containers, sothat the ratio of the reagents provides for substantial optimization ofthe method and assay. The reagents may each be in separate containers orvarious reagents can be combined in one or more containers depending onthe cross-reactivity and stability of the reagents. The kit can furtherinclude other separately packaged reagents for conducting an assayincluding ancillary reagents, and so forth.

EXAMPLES

The invention is demonstrated further by the following illustrativeexamples. Parts and percentages used herein are by weight unlessotherwise specified. Temperatures are in degrees centigrade (° C.).

Abbreviations:

Ab_(F) (anti-fluorescein)—Mouse monoclonal antibody to fluorescein.

Ab_(T3) (anti-T₃)—mouse monoclonal antibody to T₃

t-Bu—tert-butyl

TFA—trifluoroacetic acid

T₃—

Φ—chemiluminescence quantum yield

PMT—

EtoAc—ethyl acetate

BSA—Bovine serum albumin

Chl-a—Chlorophyll-a

D-H₂O—dionized water

DPP—4,7-Diphenylphenanthroline

DPPC—dipalmitoylphosphatidyl choline

DPPG—dipalmitoylphosphatidyl glycerol

DPPE—dipalmitoylphosphatidyl ethanolamine

EDAC—1-Ethyl-3-(3-Dimethylaminopropyl) carbodiimide hydrochloride.

nC₁₀—tetra-(n-decyl)phthalocyanin aluminum chloride complex.

PB—Polystyrene beads

PB/nC₁₀—PB containing nC₁₀

PBS—phosphate buffered saline 0.02M NaPi, 0.14 M NaCl/pH 7.2

Pi—Phosphate

Sulfo-NHS—Sulfo-N-hydroxysuccinimide

SATA—S-acetylthioglycolic acid N-hydroxysuccinimide ester

RLU—Relative light units.

NHS—N-hydroxysuccinimide

DMSO—dimethyl sulfoxide

DMF—dimethyl formamide

DCC—dicyclohexylcarbodiimide

TEA—triethylamine

TLC—thin layer chromatography

TNBSA—2,4,6-trinitrobenzenesulfonic acid

BGG—bovine gamma globulin

TMSCl—trimethylsilyl chloride

MeOH—methanol

Biotin-LC₇-NHS—sulfosuccinimidyl-6-(biotinamido)-hexanoate

λmax ABS—lambda maximum of absorption

λmax EMI—lambda maximum of fluorescence emission

λmax CH.EM.—lambda maximum of chemiluminescence emission

All monoclonal antibodies were produced by standard hybrid celltechnology. Briefly, the appropriate immunogen was injected into a host,usually a mouse or other suitable animal, and after a suitable period oftime the spleen cells from the host were obtained. Alternatively,unsensitized cells from the host were isolated and directly sensitizedwith the immunogen in vitro. Hybrid cells were formed by fusing theabove cells with an appropriate myeloma cell line and culturing thefused cells. The antibodies produced by the cultured hybrid cells werescreened for their binding affinity to the particular antigen, e.g. TSHor HCG. A number of screening techniques were employed such as, forexample, ELISA screens. Selected fusions were then recloned.

Example 1 Total Triiodothyronine Assay

I. Bead Preparations Materials

175 nm Carboxylate modified latex (CML beads) from Bangs Laboratories.Ethylene glycol, ethoxy ethanol, benzyl alcohol, chlorophyll-a fromAldrich.

Europium (III) thienoyl trifluoroacetonate (EUTTA) from Kodak.

Trioctyl phosphine oxide (TOPO) from Aldrich.

Dioxene [1-(4-dimethylaminophenyl)-6-phenyl 1,4 dioxene)]:

Prepared by a modification of a procedure described in: Giagnon, S. D.(1982) University Microfilms International (Ann Arbor, Mich.)

Procedures

1. Chlorophyll-a Sensitizer Beads

A solution of chlorphyll-a in benzyl alcohol (1.0 mL, 0.6 mM) was addedto 8.0 mL of benzyl alcohol at 105° C. A suspension of carboxylatemodified latex, 175 nm size, in water (10%, 1.0 mL) was added to thebenzyl alcohol solution. The mixture was stirred for 5 min at 105° C.,and cooled to room temperature. Ethanol (10.0 mL) was added and themixture centrifuged. The pellet was resuspended in a 1:1 ethanol-watermixture (10.0 mL) and the suspension centrifuged. The same resuspensionand centrifugation procedure was repeated in water (10.0 mL), and thepellet was resuspended in water (1.8 mL).

Characterization

A. Dye concentration: A solution prepared by adding 10 μL of the abovebead suspension to dioxane (990 μL) was found to have an absorbance of0.11 at 660 nm, corresponding to 2.6 μmoles of chlorophyll-a in one gramof beads.

B. Singlet oxygen generation: A mixture of chlorphyll-a beads (200 μg)2×10⁻⁴ moles of anthracene 9,10-dipropionic acid (ADPA) in two mL ofphosphate buffer (50 mM, pH 7.5, containing 100 mM NaCl) was irradiatedwith a tungsten-halogen lamp equipped with a 645 nm cut-off filter for20 min. The beads were removed by filtration, and the concentration ofthe oxygenation product was determined spectrophotometrically at 400 nm.The rate was found to be 3.0 nmoles of oxygenation product per min.Under the same conditions, 38 pmoles of a soluble sensitizer, aluminumphthalocyanin tetrasulfonate generated the same amount of oxygenationproduct (the amount of sensitizer in the beads was 200·10⁻⁶·2.6·10⁻⁶=520pmoles).

2. Chlorophyll-a/Tetrabutyl Squarate Sensitizer Beads

A suspension of carboxylated latex beads (175 nm size, 10% solids inwater, 30.0 mL) was centrifuged. The supernatant was discarded and thepellet was resuspended in ethylene glycol (60.0 mL). The suspension washeated to 100° C. 9.0 mL of a benzyl alcohol solution which is 1.67 mMin chlorophyll-a and 3.33 mM in tetrabutyl squarate [1,3bis(4-dibutylaminophenyl)squarate] was added slowly over 3 min to thesuspension. The heating was continued for 7 min, then the suspension wascooled to room temperature in a water bath. The benzyl alcoholsuspension was added to cold ethanol (120 mL). The mixture wascentrifuged and the supernatant discarded. The pellet was resuspended in50% ethanol in water and the suspension was centrifuged. The sameresuspension and centrifugation procedure was repeated in 5% ethanol inwater (30 mL).

Characterization

A. Dye concentration. The concentration of the tetrabutyl squarate inthe beads was determined spectrophotometrically as described above forthe chlorophyll-a beads. It was found to be 44 μM dye in the beads.

B. Singlet oxygen generation. Twenty-five μL of a 5 mM solution of ADPAin ethanol were added to suspension of beads (100 μg) in phosphatebuffer, pH 7.0 (20 mM, containing 50 mM NaCl). The mixture wasirradiated as above, using a 610 nm long pass filter. The rate ofsinglet oxygen formation was calculated from the rate of the decrease inabsorbance (at 400 nm) of the ADPA. It was found that the beadsgenerated 7·10⁻² μmoles of singlet oxygen/min.

3. Dioxene/EuTTA/TOPO Acceptor Beads

20 mL of 175 nm carboxylated latex beads (10% suspension in water) wasadded to ethoxy ethanol (20.0 mL). The mixture was heated to 90° C. 20mL of a solution which is 10 mM 2-(p-dimethylaminophenyl)-3-phenyldioxene, 20 mM EuTTA and 60 mM TOPO in ethoxy ethanol were added to themixture. The heating was continued for 7 min at a temperature up to 97°C. The mixture was cooled to room temperature. Ethanol (40.0 mL) wasadded and the mixture was centrifuged. The pellet was resuspended in 80%ethanol and the suspension was centrifuged. The resuspension andcentrifugation procedure was repeated in 10% ethanol (36 mL).

Characterization

A. Dye concentration. The concentration of EuTTA in the beads wasdetermined spectrophotometrically and was found to be 0.07M. Because theconcentration of dioxene cannot be determined in the presence of EuTTA,it was measured in beads which were dyed with the dioxene only,2-(p-dimethylaminophenyl)-3-phenyl dioxene, under the same conditions.The concentration was found to be 0.016M.

B. Signal generation. A suspension of beads (25 μg) in phosphate buffer(0.5 mL, 20 mM phosphate, 50 mM NaCl, 0.1% Tween 20, pH 7.0) was mixedwith a solution of 2 μM aluminum phthalocyanine tetrasulfonate (0.5 mL)in the same buffer. The mixture was illuminated for one minute with a125w tungsten-halogen lamp equipped with a 610 nm long pass filter.Following illumination, the mixture was placed in a Turner TD-20eluminometer, and the luminescence was measured for 20 sec. The intensitywas found to be 327 RLU (relative light unit)/sec. The wavelength of theemitted light was measured using Perkin-Elmer 650-40 scanningspectrofluorimeter. The major emission peak was centered near 615 nm.

II. Assay Procedure

EDAC/NHS Coupling of Antibody to 40 nm Beads

73.6 mg sulfo-NHS (N-hydroxysulfo-succinimide, Pierce Chemical Co.#24510 G) was dissolved in 6 mL of a suspension of 4 mg/mLcarboxylate-modified 40 nm polystyrene beads (dyed with chlorophyll-aand tetrabutyl squarate) in water. 136 uL 0.5 M Na₂HPO₄ was added. pHwas adjusted to 5.2. 136 uL additional water was added. 130.4 mg EDAC(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, SigmaChemical Co. #E-6383) in 454 μL water was slowly added to stirring beadsuspension. The suspension was incubated for 20 min at room temperature.The beads were centrifuged for 20 min. at 15,000 rpm in Sorvall SA-600rotor at 4° C. The supernatant was discarded. The beads were thenresuspended in 1.2 mL 5 mM sodium phosphate, pH 5.8, and the suspensionwas sonicated to redisperse beads. The beads were slowly added to 4.8 mLof a stirring solution containing 1.7 mg/mL IgG (mouse monoclonalanti-fluorescein) and 6.7 mg/mL BSA and 17 mM borax, pH 9.2, and mixedgently overnight at 4° C. 800 uL 2 M glycine was added which was thenfollowed by 2.8 mL 50 mg/mL BSA in 0.1 M borax to the bead suspension.The suspension was sonciated and allowed to mix gently for 3 h at 4° C.The beads were centrifuged for 30 min at 15,000 rpm. The supernatant wasdiscarded. The beads were resuspended in 3 mL 50 mM sodium phosphate and150 mM NaCl, pH 7.6, and the suspension was sonciated. Thecentrifugation, resuspension and sonification steps were repeated for atotal of three spins. After the third spin, beads were resuspended in2.4 mL 50 mM sodium phosphate and 150 mM NaCl, pH 7.6. The resultingsuspension was sonicated and stored at 4° C.

III. EDAC/NHS Coupling of Avidin-D to 175 nm Beads

4.4 mg sulfo-NHS was dissolved in 0.4 mL of a suspension of 25 mg/mLcarboxylate-modified 175 nm polystyrene beads (dyed with2-(p-dimethylaminophenyl)-3-phenyl dioxene/Eu(TTA)/TOPO) in water.0.0160 mL 0.25 M Na₂HPO₄ was added. 8 mg EDAC, dissolved in 0.030 mLwater, was added slowly to vortexing bead suspension. The suspension wasincubated for 20 min at room temperature. The beads were centrifuged 20min at 15,000 rpm in Sorvall SA-600 rotor at 4° C. The supernatant wasdiscarded. The beads were resuspended in 0.6 mL 0.005 M sodiumphosphate, pH 5.8. The suspension was sonicated to resuspend beads. Thebeads were again slowly added to 3 mL of a stirring solution containing1.33 mg/mL avidin-D (Vector) and 17 mM borax, pH 9.2, and mixed gentlyovernight at 4° C. 0.004 mL 1 M succinic anhydride in DMF was added .The suspension was incubated for 1 h at 4° C. with gentle mixing. 0.4 mL50 mg/mL BSA in 10 mM sodium phosphate and 150 mM NaCl, pH 7.0 wasadded. The suspension was allowed to mix gently for 3 h at 4° C. Thebeads were centrifuged for 30 min at 15,000 rpm. The supernatant wasdiscarded. The beads were resuspended in 3 mL 50 mM sodium phosphate and150 mM NaCl, pH 7.6. The suspension was sonicated. The centrifugation,resuspension and sonification steps were repeated for a total of threespins. After the third spin, the beads were resuspended in 2.25 mL 50 mMsodium phosphate and 150 mM NaCl, pH 7.6. The suspension was sonicatedand stored at 4° C.

IV. Total T₃ Assay

Assay buffer: 0.075M barbital, 0.2M NaCl, 0.4% BSA, 1.25% mouse IgG, 10mg/mL dextran sulfate (MW 500,000), 1.0 mg/mL dextran T-500, 10 μg/mLaggregated IgG.

Beads

Acceptor Beads: Avidin-EDAC, 175 nm, dyed with2-(p-dimethylaminophenyl)-3-phenyl dioxene/Eu (TTA)₃/TOPO.

Sensitizer Beads: Antifluorescein-EDAC, 40 nm, dyed withchlorophyll-a/squarate.

Assay Protocol

50 μL of 8-anilino-1-naphthalene sulfonic acid, ammonium salt (Sigma,A-3125) solution in assay buffer (0. 75 mg/mL) was added to 50 μL of T₃standard or sample. 100 μL of assay buffer was added. Biotinylatedanti-T₃ was prepared according to standard procedures by reaction ofbiotin-LC₇NHS (Pierce Chemical Company) with monoclonal anti-T₃ followedby purification by chromatography on a Sephadex column. 50 μL ofbiotinylated anti-T₃ (70 ng/mL) in assay buffer was added. The tracer,T₃—LC₂₁—Fl (1.8 ng/mL)

T₃—LC₂₁—Fl

in assay buffer (50 μL) was added. The mixture was incubated for 15minutes at 37° C. 500 μL of a suspension of sensitizer beads (50 μg) andacceptor beads (6.25 μg) in assay buffer were added, and the mixture wasincubated for 15 minutes at 37° C. The “stop solution” (50 μL) (10 μMfluorescein, 0.5 mM biotin) was added.

Signal was read by halogen lamp with a 610 nm cut-off filter, one minuteillumination, 20 sec measurement.

Results

The luminescence signal was plotted as a function of T₃ concentration.Signal modulation was 94% with 8.5 ng/mL T₃. At 0.5 ng/mL the signalmodulation was 38%.

Example 2 Chemiluminescence Quantum Yield and Decay Rate Determinations

Preparation of Compound 11

To a stirred solution of 2.55 g of 4-dimethylaminobenzoin (10 mmol) in50 mL of dry toluene, 1.2 mL of 2-mercaptoethanol (15 mmol) was added,followed by 2.5 mL of TMSCl. The reaction mixture was refluxed underargon for 18 hours, allowed to come to room temperature and poured in150 mL of saturated bicarbonate solution. The two-phase mixture wasseparated. The organic layer was again washed with 100 mL of saturatedbicarbonate solution. The combined aqueous layer was extracted with 75mL of CH₂CL₂. The combined organic layers were dried over sodium sulfate(20 g) and evaporated. The remaining residue was flash chromatographed(CH₂Cl₂) to give 2.6 g of Compounds 11 and 20 (4:1 mixture of the2-regioisomers). The ash colored solid was recrystallized fromCH₂Cl₂-MeOH (10:90) mixture to yield 1.8 g of needle-shaped crystals ofa single regioisomer of compound 11.

M.P. 108-110° C. ¹HNMR (CDCl₃, 250 MHz): δ 2.85 (s, 6H), 3.22 (t, 2H),4.5 (t, 2H), 6.55 (d, 2H), 7.1-7.3 (m, 7H).

Mass Spectrum (CI: m/e, relative intensity) Major Peaks: 297 (M⁺, 40),165 (100).

Absorption Spectra (Toluene): 330 nm (ε 13,000).

Photooxygenation Procedure

25 Milligrams of Compound 11 (major regioisomer from above) wasdissolved in 10 mL of CH₂Cl₂ in a photooxygenation tube. Approximately50 mg of polystyrene bound Rose Bengal was added and oxygen bubblerconnected. Oxygen was passed slowly through the solution while thesample as irradiated with a Dolan-Jenner lamp equipped with a 500 nmcut-off filter. Progress of the reaction was monitored by TLC. A spotfor the thioester product could be detected and had a lowerR_(f)(CH₂Cl₂) than Compound 11. The reaction was judged complete whenCompound 13 was completely consumed. The sensitizer was filtered off andsolution was evaporated on a rotary evaporator to yield 26 mg ofthioester 32 as the only product.

1HNMR: (CD₂CL₂): δ 3.05 (s, 6H), 3.4 (5, 2H), 4.45 (5, 2H), 6.72 (d,2H), 7.5 (m, 3H), 7.85 (d, 2H), 8.05 (d, 2H).

Mass Spectra (CI, relative intensity) Major Peaks: 329 (M⁺, 25), 148(100).

Absorption Spectrum (CH₂Cl₂): 342 nm (˜30,000).

Fluorescence Spectrum (Toluene): 370 nm.

Fluorescence Measurements

A solution of thioester 32 was taken in four different solvents(Toluene-dry; CH₂Cl₂; hexane; and acetonitrile) and placed in a 1-cmsquare quartz cuvette in the sample compartment of a Perkin-Elmer 650-40fluorometer. The sample was excited at the absorption maxima of eachsolvent (slit width 2 nm) and emission spectra (slit width 3 nm) wasrecorded by scanning from 350 nm to 470 nm. The fluorescence efficiencywas determined and tabulated in Table 1.

TABLE 1 Efficiency of Thioester in Different Solvents λ λ ABS EMICompound Solvent nM nM Φ Diester* Toluene 314 360 0.1 400 Thioester 32**Toluene 338 370 0.025 CH₂Cl₂ 340 390 0.07 Hexane 332 370 ˜0.006 CHCN 342390 ˜0.006 *2-(p-dimethylaminophenyl)3-phenyl ethyldiester, Giagnon,S.D. (1982) University Microfilms International (Ann Arbor, Michigan).**Thioester is rapidly photobleached on excitation at 340 nm in toluene.

Determination of Quantum Yield of Chemiluminescence.

Preparation of Eu(TTA)Phen:

8.69 g of Eu(TTA)₃. 3H₂O (10 mmoles, Kodak) and 1.8 g of1,10-phenanthroline (10 mmoles, Aldrick) in 50 ml of dry toluene wereheated to 95° C. in an oil bath for one 1 hour. Toluene was removedunder reduced pressure. The ash coloured solid was cystallized from 10ml of toluene to yeild 10 grams of Eu(TTA)₃Phen.

Absorption spectrum: 270 nm (20,000), 340 nm (60,000) (Toluene);1.R(KBr): Cm⁻¹: 3440(s), 1600(8), 1540(s), 1400(s), 1300(s).

Energy Transfer to Eu(TTA)₃Phen

A solution of Compound 11 (regioisomers from above) (0.1 mM) (8:2mixture), aluminum phthalocyanine (0.1 μM), and Eu(TTA)₃Phen from above(0-4.0 mM) in dry toluene was placed in a 1-cm square quartz cuvette(two sided silvered) in the sample compartment of a Spex Fluorologspectrophotometer. The temperature of the sample holder was maintainedby a circulating external water bath at 25° C. A 640 nm cut-off filterwas placed in front of the excitation beam. The sample solutions wereplaced in the sample compartment for at least 3 minutes for thermalequilibrium to be reached. The emission was recorded in the time drivemode. Samples were irradiated at 680 nm (slit width 24 nm) until asteady state of emission at 613 nm (slit width 8 nm) was reached. Thesteady state light intensity at various concentrations of Eu(TTA)₃Phenwas recorded and is summarized in Table 2. From the steady state lightintensity quantum yields were determined. Double reciprocal plots ofchemiluminescence intensity against Eu(TTA)₃Phen concentration werelinear.

TABLE 2 Chemiluminescence Efficiency as a Function of Eu(TTA)₃ PhenConcentration Compound 11* Eu(TTA)₃Phen mM mM RLU at 613 nm 0.1 0 — 0.10.05 7.43 × 10⁴ 0.1 0.1  1.8 × 10⁵ 0.1 0.2 2.89 × 10⁵ 0.1 0.5 6.13 × 10⁵0.1 1.0 9.45 × 10⁵ 0.1 2.0 1.17 × 10⁶ 0.1 4.0 1.32 × 10⁶ 0.1** 4.0  1.6× 10⁶ *Except for last run Compound 11 used in these experimentscontained 20% of its regioisomer 20 **Compound 11 used in thisexperiment was greater than 98% of a single regioisomer.

Chemiluminescence from Dioxene 9

Experiment 1: A solution of dioxene 9 (0.1 mM) and aluminumphthalocyanine (0.1 μM) in dry toluene was irradiated at 680 nm asdescribed above. The emission in light intensity at 400 nm (slit width 8nm) was recorded as a function of irradiation time. The light intensitywas 8793 RLU's for 180 seconds of irradiation (average of threeexperiments).

Experiment 2: Rate of dioxene 9 dioxetane decomposition was monitored bydecay of chemiluminescence of an aerated solution in dry toluene at 25°C. Rate of decomposition was monitored in the presence of 1.0 μMaluminum phthalocyanine and dioxene (less than 0.1 mM of dioxene). Thechemiluminescence decay was monitored on Spex Fluorologspectrophotometer under previously described conditions. The rateconstant of decay at 25° C. was 2.88×10⁻⁴ S⁻¹.

Preparation of Acceptor Beads

Four mL of 20% suspension (400 mg) of washed 175 nm carboxylate modifiedlatex was diluted with 3 mL of ethoxyethanol in a 25 mL round bottom(R.B.) flask with a stir bar. The R.B. flask was then placed in an oilbath at 105° C. and stirred for 10 minutes. Then, 3.3 mM thioxene 11 and15.5 mM Eu(TTA)₃DPP was added; the beads were stirred for 5 minutesmore. At this point 1.0 mL of 0.1N NaOH was added slowly over 5 minutes.During all the additions, the oil bath temperature was maintained at105° C. The oil bath temperature was slowly allowed to drop to roomtemperature over 2 hours.

After cooling, the mixture was diluted with 20 mL of ethanol andcentrifuged (12,500 rpm, 30 minutes). Supernatants were discarded andthe pellets resuspended in ethanol by sonication. Centrifugation wasrepeated, and the pellet was resuspended in water; and centrifugationwas repeated. The pellet was resuspended in 5 mL of aqueous ethanol to afinal volume of 40 mL. The final concentration of the beads was 10mg/mL.

The concentration of Eu(TTA)₃DPP was determined spectrophotometrically.An aliquot of the bead suspension was reduced to dryness under a streamof dry argon and the residue dissolved in dioxane. Using a density of1.06 g/cc for polystyrene, (ε 340 nm=6.7×10⁴) for Eu(TTA)₃ and (ε 270nm=4.0×10⁴) for DPP, the concentration of Eu(TTA)₃DPP was determined tobe 100 mM. The concentration of compound 11 in the beads could not bedetermined because its absorbance was masked by Eu(TTA)₃DPP.

Chemiluminescence of the beads was measured in an ORIEL luminometerusing water-soluble aluminum phthalocyanine sensitizer. An aliquot ofbeads was diluted to 100 μg/mL in phosphate buffer pH 8.0 containing0.1% Tween-20. 1.0 μM of aluminum phthalocyanine tetrasulfonic acid wasadded and chemiluminescent signal was measured as a function ofirradiation time. An identical sample was also placed in a SpexFluorolog fluorometer and irradiated at 680 nm (slit width 20 nm; 640cut-off filter). The chemiluminescence emission spectra was recorded byscanning from 570 nm to 620 nm. Chemiluminescence decay and quantumyields is summarized in Table 3.

Determination of Quantum Yields in Beads

Dioxene 9 Beads

A solution of dioxene 9 beads (0.2 mg) and aluminum phthalocyaninetetrasulfonic acid (2.5 μM) in phosphate buffer (pH 8.2; 50 mM 0.1%Tween-20) was placed in a 1 cm quart cuvette (two sides silvered) in thesample compartment of a Spex Fluorolog spectrophotometer. Thetemperature of the sample holder was maintained 25° C. A 640 cut-offfilter was placed in front of the excitation beam. The sample solutionswere placed in the sample compartment for at least 3 minutes for thermalequilibrium to be reached. The light emission at 360 nm was followed inthe time drive mode. Samples were irradiated at 680 nm (slit width 24nm) for 60 seconds. The emission at 360 nm (slit width 16 nm) wasrecorded with time for 5000 seconds. Total light emitted was determinedby the cut-weigh method. Peak shape correction was also done by the cutand weigh method. The total light emitted at 360 nm was 8.87±0.2×10⁴RLU's/4500 seconds (after peak shape correction; average of 2experiments).

Dioxene 9: Eu(TTA)₃TOPO Beads

A solution of dioxene 9 Eu(TTA)₃TOPO beads (0.2 mg) and aluminumphthalocyanine tetrasulfonic acid (2.5 μM) in phosphate buffer (pH 8.2,50 mM 0.1% Tween-20) was placed in a 1-cm quartz cuvette (two sidessilvered) in the sample compartment of a Spex Fluorologspectrophotometer. The rest of the experiment was performed as describedfor dioxene 9 beads. The light emission from beads was followed at 613nm (slit width 16 nm).

Total light emitted was determined by the cut and weigh method. PMTcorrection was done as described previously in solution studies. Thetotal light emitted at 613 nm was 25.0±0.3×10⁵ RLU's/4500 seconds (afterPMT correction; average of 2 experiments)

Steady State Methods

Dioxene 9: Eu(TTA)₃TOPO Beads. A solution of dioxene 9: Eu(TTA)₃TOPObeads (0.5 mg) and aluminum phthalocyanine tetrasulfonic acid (0.05 μM)in phosphate buffer (pH 8.2; 50 mM 0.1% Tween-20) was placed in a 12-75mM test tube in the sample compartment of an Oriel chemiluminometer. Thetemperature of the sample holder is 37° C. A 610 cut-off filter wasplaced in front of the excitation beam. The sample solutions were placedin the sample compartment for at least 5 minutes for thermal equilibriumto be reached. The sample was irradiated for 30-second intervalsfollowed by a 5-second read time until a steady state of emission isreached. The average intensity at steady state emission is 21,000±1000RLU's (3 experiments).

Compound 11: Eu(TTA)₃DPP Beads. A solution of thioxene 11: Eu(TTA)₃DPPbeads (0.5 mg) and aluminum phthalocyanine tetrasulfonic acid (0.05 μM)in phosphate buffer (pH 8.2, 50 mM, 0.1% Tween-20) was placed in 12-75mM test tube in the sample compartment of an Oriel chemiluminometer. Thetemperature of the sample holder is 37° C. A 610 cut-off filter wasplaced in front of the excitation beam. The sample solutions were placedin the sample compartment for at least 5 minutes for thermal equilibriumto be reached. The sample was irradiated for 6-second intervals followedby 3 seconds read time until a steady state of emission was reached. Theaverage intensity at steady state emission is 32,000±1000 (3experiments).

TABLE 3 Chemiluminescent Properties of Thioxene 11 and Dioxene 9 λmaxCompound Medium (CH.EM) t½ Φ 11 Toluene 400 nM 2.1 sec low* (100 μM)11 + Toluene 613 nM 1.8-2.1 sec 0.20*** Eu(TTA)₃Phen (4 mM) 11 + CMLbeads 613 nM decay 0.46 Eu(TTA)₃DPP multiphasic (100 mM) (initial t½ at37° C. is −0.5 secs) 9 Toluene 420 nM 3462 sec 0.015 (100 μM) ** CMLbeads 360 nM decay 0.008 multiphasic 9 + CML beads 613 nM decay 0.31Eu(TTA)₃ • TOPO (Major) multiphasic (16 mM) 400 nM *less than 0.0003**Control, no compound present ***0.37 for Eu(TTA)₃Phen concentrationextrapolated to infinity

Example 3

Preparation of C-26 Thioxene (Compound 13):

A. 62 g of N-methyl aniline (0.5 mole) and 62 g of ethyl 5-bromovalerate(0.3 mole) were heated to 100° C. in a sealed tube for 16 hours. Thereaction mixture was cooled to room temperature and poured into 100 mlof ethyl acetate. The ethyl acetate solution was washed with 20% sodiumhydroxide (3×100 ml). The aqueous layer was extracted with 50 ml ofethyl acetate. The combined ethyl acetate solution was dried over sodiumsulphate (50 g) and removed under reduced pressure. The residue wasdistilled under high vacuum (130-137° C.) to yield 60 g of N-methylN-ethyl valerate aniline.

¹H NMR (CDCl₃, 250 MHz): δ 1.3 (t, 3H), 1.65 (m, 4H), 2.3 (t, 2H), 2.8(s, 3H), 3.3 (t, 2H), 4.2 (q, 2H), 6.65 (d, 2H), 7.2 (m, 3H).

B. To a stirred solution of DMF (8.8 g) in an ice bath POCl₃ (5.06 g)was added slowly. After the addition was complete, the reaction isstirred at 4° C. for 10 minutes. N-methyl N-ethyl valeroyl aniline fromPart A above (3.76 g) was added and the reaction was heated to 100° C.for 1 hour. The reaction mixture was poured into ice and neutralizedwith 20% sodium hydroxide. The mixture was extracted with ethyl acetate(3×50 ml). The combined ethyl acetate solution was dried over sodiumsulphate (50 g) and removed under reduced pressure. The residue waspassed through silica gel (CH₂Cl₂→CH₂Cl₂: EtOAc 9:2).

¹H NMR (CDCl₃, 250 MHz): δ 1.2 (t, 2H), 1.6 (m, 4H), 2.3 (t, 2H), 2.9(s, 3H) 3.3 (t, 2H), 4.1 (q, 2H), 6.6 (d, 2H), 7.6 (d, 2H), 9.7 (s, 1H).

C. To a refluxing solution of 5.Og of N-methyl

N-ethyl-ω-valeroyl p-formyl aniline from Part B above (20 mmole) and 2 gof potassium cyanide in 60% ethanol under argon was added 2.15 g ofbenzaldehyde (20 mmole) in 20 ml of ethanol in 90 minutes. The reactionmixture was refluxed for 15 minutes more and extracted with ethylacetate (3×50 ml). The combined ethyl acetate solution was dried oversodium sulphate (50 g) and removed under reduced pressure. The productwas purified on preparative TLC (hexane: ethyl acetate 5:1) to yield 2.2g of substituted benzoin.

¹H NMR (CDCl₃, 250 MHz): δ 1.3 (t, 3H), 1.6 (m, 4H), 2.4 (t, 2H), 2.9(s, 3H), 3.3 (t, 2H), 4.1 (q, 2H), 4.8 (d, 1H), 5.8 (d, 1H), 6.5 (d,2H), 7.3 (m, 5H), 7.8 (d, 2H).

D. To a stirred solution of the benzoin from Part C above (1.1 g) in 15ml of ethanol was added 7 ml of water and 100 mgs of KOH. The reactionwas stirred at room temperature for 3 hours. TLC (silica gel , CH₂Cl₂:EtOAc 9:1) showed no starting material. The solvent was neutralized andthe carboxylic acid product was extracted with ethyl acetate (5×50 ml).The combined ethyl acetate solution was dried over sodium sulphate (50g) and removed under reduced pressure. The carboxylic acid product wasused as is for the next step.

¹H NMR (CDCl₃, 250 MHZ): δ 1.6 (m, 4H), 2.4 (t, 2H), 2.9 (s, 3H), 3.3(t, 2H), 5.8 (s, 1H), 6.5 (d, 2H), 7.3 (m, 5H), 7.8 (d, 2H).

E. To a stirred solution of the carboxylic acid from Part D above (1.7g, 5 mmole) and didecyl amine (1.9 g, 6.3 mmole) in 80 ml of DMF at 4°C. was added DPPA (1.8 g, 8 mmole) followed by addition of triethylamine (1.25 ml). The reaction mixture was stirred at 4° C. and then atroom temperature for 16 hours. The solvent was neutralized and theproduct was extracted with ethyl acetate (5×50 ml). The combined ethylacetate solution was dried over sodium sulphate (50 g) and removed underreduced pressure. The product was purified on preparative TLC (CH₂Cl₂:ethyl acetate 9:1) to yield 2.6 g of substituted amide benzoin.

¹H NMR (CDCl₃, 250 MHz): δ 0.8 (t, 6H), 1.3 (m, 36H), 1.6 (m, 12H), 2.3(t, 2H), 2.7 (m, 4H), 3.0 (s, 3H), 3.3 (m, 6H), 4.8 (d, 1H), 5.8 (d,1H), 6.5 (d, 2H), 7.3 (m, 5H), 7.8 (d, 2H).

F. To a stirred solution of 1.5 g of substituted benzoin (2.5 mmole) in50 ml of dry toluene, 1.2 ml of 2-thioethanol (15 mmole) was added,followed by 2.5 ml of TMSCl. The reaction mixture was refluxed in an oilbath under argon for 30 hours. The reaction mixture was allowed to cometo room temperature poured into 150 ml of saturated bicarbonatesolution. The organic layer was separated and washed with 100 ml ofsaturated bicarbonate solution. The combined aqueous layer was extractedwith 75 ml of CH₂Cl₂.

The combined organic solution was dried over sodium sulphate (50 g) andremoved under reduced pressure.The product was purified on silica gel(CH₂Cl₂: ethyl acetate 9:1) to yield 1.2 g of C-26 thioxene as an paleyellow oil.

¹H NMR (CDCl₃, 250 MHz): δ 0.8 (t, 6H), 1.3 (m, 36H), 1.6 (m, 12H), 2.3(t, 2H), 2.8 (s, 3H), 3.3 (m, 9H), 4.5 (t, 2H), 6.5 (d, 2H), 7.1 (d,2H), 7.3 (m, 5H). Mass Spectrum (CI: m/e) M⁺ 662. Absorption Spectra(Toluene): 330 nm (ε 13,000).

Example 4 Preparation of C-8 Thioxene (Compound 15)

A. To a refluxing solution of 3.0 g of p-dimethylamino-benzaldehyde (20mmole) and 2 g of potassium cyanide in 60% ethanol under argon was added4.4 g of p-octyl benzaldehyde (20 mmole, Kodak) in 20 ml of ethanol in90 minutes. The reaction mixture was refluxed for 15 minutes more andextracted with ethyl acetate (3×50 ml). The combined ethyl acetatesolution was dried over sodium sulphate (50 g) and removed under reducedpressure. The product was purified on preparative TLC (hexane: ethylacetate 5:1) to yield 1.2 g of substituted benzoin.

¹H NMR (CDCl₃, 250 MHz): δ 0.85 (t, 3H), 1.3 (m, 12H), 1.5 (m, 2H), 2.5(t, 2H), 2.9 (s, 6H), 4.8 (d, 1H), 5.8 (d, 1H), 6.5 (d, 2H), 7.3 (q,4H), 7.8 (d, 2H).

B. To a stirred solution of 0.94 g of substituted benzoin from Part Aabove (2.5 mmole) in 50 ml of dry toluene, 1.2 ml of 2-thioethanol (15mmole) was added, followed by 2.5 ml of TMSCl. The reaction mixture wasrefluxed in an oil bath under argon for 30 hours. The reaction mixturewas allowed to come to room temperature and poured into 150 ml ofsaturated bicarbonate solution. The organic layer was separated andwashed with 100 ml of saturated bicarbonate solution. The combinedaqueous layer was extracted with 75 nl of CH₂Cl₂. The combined organicsolution was dried over sodium sulphate (50 g) and removed under reducedpressure. The product was purified on silica gel (CH₂Cl₂: ethyl acetate9:1) to yield 0.75 g of C-8 thioxene Compound 15 as pale yellow solid.

¹H NMR (CDCl₃, 250 MHz): δ 0.8 (t, 3H), 1.3 (m, 10H), 1.6 (m, 2H), 2.5(t, 2H), 2.9 (s, 6H), 3.3 (t, 2H), 4.5 (t, 2H), 6.5 (d, 2H), 7.1 (d,2H), 7.3 (m, 5H). Mass Spectrum (CI: m/e, relative intensity) 409(M⁺100), 165 (40). Absorption Spectra (Toluene): 330 nm (ε 13,000).

Example 5 Preparation of N-Phenyl Oxazine (Compound 16)

A. 5 g of p-dimethylaminobenzoin was dissolved in 5 ml of CH₂Cl₂ andstirred in an ice bath. 10 ml of SOCl₂ was added and the reactionmixture stirred for 1 hour. The solvent was removed under reducedpressure and the product was crystallized from MeOH.

¹H NMR (CDCl₃, 250 MHz): δ 3.0 (s, 6H), 6.3 (s, 1H), 6.5 (d, 2H), 7.4(m, 5H) 7.8 (d, 2H).

B. 0.271 g of P-(2-phenyl-2-chloro acetyl) dimethylamino-benzene (1mmole) and 0.274 g of N-(2 hydroxy ethyl) aniline (2.0 mmole) weredissolved in 3 ml of dry ethanol and heated in a sealed tube at 80° C.for 8 hours. On cooling the product crystallized out as pale yellowneedles, which was filtered and dried to yield 0.2 g of N-phenyl oxazineCompound 15.

1H NMR (CDCl₃, 250 MHz): δ 3.0 (bs, 6H), 3.7 (bt, 2H), 4.4 (bt, 2H), 6.5(bd, 2H), 7.4 (m, 12H). Mass Spectrum (CI: m/e, relative intensity) 356(M⁺, 100), 180 (70).

Example 6 Preparation of N-Phenyl Indole Oxazine (Compound 17)

0.283 g of 3-(2-phenyl-2-chloro acetyl) N-methylindole (1 mmole) (H.Nakamura and T. Goto, Heterocyles, 10, 167-170 (1978). and 0.274 g of2-anilino ethanol (2.0 mmole) were dissolved in 3 ml of dry ethanol andheated in a sealed tube at 80° C. for 8 hours. On cooling the productcrystallized out as pale yellow needles which was filtered and dried toyield 0.21 g of N-phenyl indole oxazine Compound 17.

1H NMR (CDCl₃, 250 MHz): δ 3.7 (bs, 3H), 3.8 (bt, 2H), 4.4 (bt, 2H), 7.2(bm, 15H). Mass Spectrum (CI: m/e, relative intensity) 366 (M⁺100), 180(70).

Example 7

Table 4 summarizes the properties of Compounds 11, 16, and 17 determinedin a manner similar to that described in Example 2.

TABLE 4 Properties of Chemiluminescent Compounds And Compositions λmaxλmax λmax Compound** (AbS) (EMI) (CH.EM) t½ Φ 11 330 nM 400 nM 400 nM2.1 sec Low* (b) 11 + 615 nM 1.3 sec 0.0024 Eu(TTA)₃ (a)(b) 11 + 615 nM1.8 sec 0.14 Eu(TTA)₃Phen (a)(b) 16 400 nM 550 nM 120 sec  Low* (b) 16 +Eu(TTA)₃ 615 nM  11 sec 0.005 (1.5 × 10⁻⁴M) 16 + Eu(TTA)₃ 615 nM 3.5 sec0.04 (5.0 × 10⁻⁴M) (b)(c)(d) 17 550 nM 120 sec  Low* 17 + Eu(TTA)₃ 615nM  12 sec 0.04 (0.6 × 10⁻⁴M) 17 + Eu(TTA)₃ 615 nM   2 sec 0.026 (0.6 ×10⁻⁴M) (b)(c)(d) *less than 0.0003 **in toluene

a) R¹O₂— Rate of reaction of singlet oxygen with thioxene in toluene is18.9×10⁷ M⁻¹ sec⁻¹. After correction for regioisomers and rate ofreaction, the quantum yield was determined.

b) Quantum yield determined by steady state method.

c) Assuming that the rate of reaction of singlet oxygen with morphilinooxene and dioxene is the same.

d) The rate of chemiluminescence decay and quantum yield depend onEu(TTA)₃ concentration.

The above discussion includes certain theories as to mechanisms involvedin the present invention. These theories should not be construed tolimit the present invention in any way, since it has been demonstratedthat the present invention achieves the results described.

The above description and examples disclose the invention includingcertain preferred embodiments thereof. Modifications of the methodsdescribed that are obvious to those of ordinary skill in the art areintended to be within the scope of the following claims and includedwithin the metes and bounds of the invention.

What is claimed is:
 1. A method for determining an analyte whichcomprises: (a) providing in combination (1) a medium suspected ofcontaining an analyte, (2) a photosensitizer capable in its excitedstate of activating oxygen to a singlet state, said photosensitizerassociated with a first specific binding pair (sbp) member, and (3) acomposition comprising a latex particulate material having incorporatedtherein a composition comprising: (i) a metal chelate comprising a metalselected from the group consisting of europium, terbium, dysprosium,samarium, osmium and ruthenium in at least a hexacoordinated state and(ii) a compound having the structural portion:

 wherein X is O, S or N and the valency of N is completed with hydrogenor an organic radical consisting of atoms selected from the groupconsisting of C, O, N, S, and P and Ar and Ar′ are independently aryland one of Ar or Ar′ is electron donating with respect to the other, andwherein the broken lines are to hydrogen, an organic radical or aretaken together to form a ring, said compound being capable of undergoinga chemical reaction with singlet oxygen to form a metastableintermediate that can decompose with the emission of light within thewavelength range of 250 to 1200 nm, wherein said latex particulatematerial has bound thereto a second sbp member that is capable ofbinding directly or indirectly to said analyte or to said first sblmember, (b) treating said combination with light to excite saidphotosensitizer, and (c) examining said combination for the amount ofluminescence emitted therefrom, the amount of said luminescence beingrelated to the amount of analyte in said medium.
 2. A kit comprising inpackaged combination: (a) a composition comprising a latex particulatematerial having incorporated therein a composition comprising: (i) ametal chelate comprising a metal selected from the group consisting ofeurorium, terbium, dysgrosium, samarium, osmium and ruthenium in atleast a hexacoordinated state and (ii) a compound having the structuralportion:

 wherein X is O, S or N and the valency of N is completed with hydrogenor an organic radical consisting of atoms selected from the groupconsisting of C, O, N, S, and P and Ar and Ar′ are independently aryland one of Ar or Ar′ is electron donating with respect to the other, andwherein the broken lines are to hydrogen, an organic radical or aretaken together to form a ring, said compound being capable of undergoinga chemical reaction with singlet oxygen to form a metastableintermediate that can decompose with the emission of light within thewavelength range of 250 to 1200 nm, wherein said latex particulatematerial has bound thereto an sbp member, and (b) a photosensitizer thatis not in said composition and is capable in its excited state ofactivating oxygen to its singlet state.
 3. A method for determining ananalyte which comprises: (a) providing in combination (1) a mediumsuspected of containing an analyte, (2) a photosensitizer capable in itsexcited state of activating oxygen to a singlet state, saidphotosensitizer associated with a specific binding pair (sbp) member,and (3) a suspendible latex particulate material comprising achemiluminescent compound, said particulate material having boundthereto an sbp member, said chemiluminescent compound having theformula:

wherein X′ is S or NR′ wherein R′ is alkyl or aryl and D and D′ areindependently selected from the group consisting of alkyl and alkylradical, (b) treating said combination with light to excite saidphotosensitizer, and (c) examining said combination for the amount ofluminescence emitted therefrom, the amount of said luminescence beingrelated to the amount of analyte in said medium.
 4. The method of claim3 wherein R′ is methyl or phenyl.
 5. The method of claim 3 wherein saidparticulate material comprises a metal chelate comprising a metalselected from the group consisting of europium, terbium, dysprosium,samarium, osmium and ruthenium in at least a hexacoordinated state.
 6. Amethod for determining an analyte which comprises: (a) providing incombination (1) a medium suspected of containing an analyte, (2) aphotosensitizer capable in its excited state of activating oxygen to asinglet state, said photosensitizer associated with a first specificbinding pair (sbp) member, (3) a composition comprising a suspendiblelatex particulate material having incorporated therein a compositioncomprising (i) a metal chelate comprising a metal selected from thegroup consisting of europium, terbium, dysprosium, samarium, osmium andruthenium in at least a hexa coordinated state, and (ii) achemiluminescent compound, said particulate material having boundthereto a second sbp member that is capable of binding directly orindirectly to said analyte or to said first sbp member, saidchemiluminescent compound having the formula:

wherein X′ is S or NR′ wherein R′ is alkyl or aryl and D and D′ areindependently selected from the group consisting of alkyl and alkylradical (b) treating said combination with light to excite saidphotosensitizer, and (c) examining said combination for the amount ofluminescence emitted therefrom, the amount of said luminescence beingrelated to the amount of analyte in said medium.
 7. The method of claim6 wherein R′ is methyl or phenyl.
 8. The compound of claim 6 whereinalkyl is (CH₂)₆CH₃ or (CH₂)₇CH₃.